Management of pyruvate kinase deficiency in children and adults.

Pyruvate kinase deficiency (PKD) is an autosomal-recessive enzyme defect of the glycolytic pathway that causes congenital nonspherocytic hemolytic anemia. The diagnosis and management of patients with PKD can be challenging due to difficulties in the diagnostic evaluation and the heterogeneity of clinical manifestations, ranging from fetal hydrops and symptomatic anemia requiring lifelong transfusions to fully compensated hemolysis. Current treatment approaches are supportive and include transfusions, splenectomy, and chelation. Complications, including iron overload, bilirubin gallstones, extramedullary hematopoiesis, pulmonary hypertension, and thrombosis, are related to the chronic hemolytic anemia and its current management and can occur at any age. Disease-modifying therapies in clinical development may decrease symptoms and findings associated with chronic hemolysis and avoid the complications associated with current treatment approaches. As these disease-directed therapies are approved for clinical use, clinicians will need to define the types of symptoms and findings that determine the optimal patients and timing for initiating these therapies. In this article, we highlight disease manifestations, monitoring approaches, strategies for managing complications, and novel therapies in development.

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 6 months 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.

Molecular genetic testing enabled the diagnosis of otherwise undiagnosable cases of pyruvate kinase deficiency.

The gold standard for the diagnosis of pyruvate kinase (PK) deficiency, the most frequent red blood cell enzymopathy, is an enzymatic activity assay. However, this assay is rather unreliable in a clinical setting, often leading to misdiagnosis or missed diagnosis. This report presented the cases of two patients diagnosed with PK deficiency using molecular genetic testing, even though conventional laboratory tests, including the PK activity assay, failed to detect any abnormalities. Genetic analysis of the patients and their asymptomatic parents revealed the presence of variants in both alleles of the PKLR gene that were assessed as "likely pathogenic" or "pathogenic" in the form of compound heterozygotes. One of the mutations detected was common in both patients. Our results suggested that genetic testing might be required for the reliable diagnosis of suspected congenital hemolytic anemia cases displaying atypical presentation.

Improving the laboratory diagnosis of pyruvate kinase deficiency.

Pyruvate kinase (PK) deficiency is an autosomal recessive disease caused by mutations in the PKLR gene, which reduce erythrocyte PK enzyme activity and result in decreased energy synthesis in red cells, causing haemolytic anaemia. Historically, the investigation into pyruvate kinase deficiency (PKD) has been led by a red cell enzyme assay determining PK enzyme activity per unit of haemoglobin. For our laboratory, the reference range was set by Beutler et al. in 1977 when the test was first established. The introduction of genetic testing permitted the creation of reference sample datasets, with positive controls having two pathogenic variants causing disease. This permitted re-assessment of the enzyme assay's sensitivity and specificity, and was used to reassess the reference range of the enzyme assay. Using sequenced samples, we have devised an enzyme assay, DNA testing workflow, which minimises false negative/positive results and improves the diagnostic efficiency. This combined enzyme-DNA testing strategy should improve the diagnostic accuracy whilst limiting the number of expensive DNA tests. During this evaluation, 10 novel genetic variants were identified and are described.

Untargeted metabolic profiling in dried blood spots identifies disease fingerprint for pyruvate kinase deficiency.

The diagnostic evaluation and clinical characterization of rare hereditary anemia (RHA) is to date still challenging. In particular, there is little knowledge on the broad metabolic impact of many of the molecular defects underlying RHA. In this study we explored the potential of untargeted metabolomics to diagnose a relatively common type of RHA: Pyruvate Kinase Deficiency (PKD). In total, 1903 unique metabolite features were identified in dried blood spot samples from 16 PKD patients and 32 healthy controls. A metabolic fingerprint was identified using a machine learning algorithm, and subsequently a binary classification model was designed. The model showed high performance characteristics (AUC 0.990, 95%CI 0.981-0.999) and an accurate class assignment was achieved for all newly added control (13) and patient samples (6), with the exception of one patient (accuracy 94%). Important metabolites in the metabolic fingerprint included glycolytic intermediates, polyamines and several acyl carnitines. In general, the application of untargeted metabolomics in dried blood spots is a novel functional tool that holds promise for diagnostic stratification and studies on disease pathophysiology in RHA.

Pyruvate kinase deficiency in children.

BACKGROUND: Pyruvate kinase deficiency (PKD) is a rare, autosomal recessive red blood cell enzyme disorder, which leads to lifelong hemolytic anemia and associated complications from the disease and its management. METHODS: An international, multicenter registry enrolled 124 individuals younger than 18 years old with molecularly confirmed PKD from 29 centers. Retrospective and prospective clinical data were collected. RESULTS: There was a wide range in the age at diagnosis from 0 to 16 years. Presentation in the newborn period ranged from asymptomatic to neonatal jaundice to fulminant presentations of fetal distress, myocardial depression, and/or liver failure. Children <5 years old were significantly more likely to be transfused than children >12 to <18 years (53% vs. 14%, p = .0006), which correlated with the timing of splenectomy. Regular transfusions were most common in children with two severe PKLR variants. In regularly transfused children, the nadir hemoglobin goal varied considerably. Impact on quality of life was a common reason for treatment with regular blood transfusions and splenectomy. Splenectomy increased the hemoglobin and decreased transfusion burden in most children but was associated with infection or sepsis (12%) and thrombosis (1.3%) even during childhood. Complication rates were high, including iron overload (48%), perinatal complications (31%), and gallstones (20%). CONCLUSIONS: There is a high burden of disease in children with PKD, with wide practice variation in monitoring and treatment. Clinicians must recognize the spectrum of the manifestations of PKD for early diagnostic testing, close monitoring, and management to avoid serious complications in childhood.

Clinical spectrum of pyruvate kinase deficiency: data from the Pyruvate Kinase Deficiency Natural History Study.

An international, multicenter registry was established to collect retrospective and prospective clinical data on patients with pyruvate kinase (PK) deficiency, the most common glycolytic defect causing congenital nonspherocytic hemolytic anemia. Medical history and laboratory and radiologic data were retrospectively collected at enrollment for 254 patients with molecularly confirmed PK deficiency. Perinatal complications were common, including anemia that required transfusions, hyperbilirubinemia, hydrops, and prematurity. Nearly all newborns were treated with phototherapy (93%), and many were treated with exchange transfusions (46%). Children age 5 years and younger were often transfused until splenectomy. Splenectomy (150 [59%] of 254 patients) was associated with a median increase in hemoglobin of 1.6 g/dL and a decreased transfusion burden in 90% of patients. Predictors of a response to splenectomy included higher presplenectomy hemoglobin (P = .007), lower indirect bilirubin (P = .005), and missense PKLR mutations (P = .0017). Postsplenectomy thrombosis was reported in 11% of patients. The most frequent complications included iron overload (48%) and gallstones (45%), but other complications such as aplastic crises, osteopenia/bone fragility, extramedullary hematopoiesis, postsplenectomy sepsis, pulmonary hypertension, and leg ulcers were not uncommon. Overall, 87 (34%) of 254 patients had both a splenectomy and cholecystectomy. In those who had a splenectomy without simultaneous cholecystectomy, 48% later required a cholecystectomy. Although the risk of complications increases with severity of anemia and a genotype-phenotype relationship was observed, complications were common in all patients with PK deficiency. Diagnostic testing for PK deficiency should be considered in patients with apparent congenital hemolytic anemia and close monitoring for iron overload, gallstones, and other complications is needed regardless of baseline hemoglobin. This trial was registered at www.clinicaltrials.gov as #NCT02053480.

Molecular heterogeneity of pyruvate kinase deficiency.

Red cell pyruvate kinase (PK) deficiency is the most common glycolytic defect associated with congenital non-spherocytic hemolytic anemia. The disease, transmitted as an autosomal recessive trait, is caused by mutations in the PKLR gene and is characterized by molecular and clinical heterogeneity; anemia ranges from mild or fully compensated hemolysis to life-threatening forms necessitating neonatal exchange transfusions and/or subsequent regular transfusion support; complications include gallstones, pulmonary hypertension, extramedullary hematopoiesis and iron overload. Since identification of the first pathogenic variants responsible for PK deficiency in 1991, more than 300 different variants have been reported, and the study of molecular mechanisms and the existence of genotype-phenotype correlations have been investigated in-depth. In recent years, during which progress in genetic analysis, next-generation sequencing technologies and personalized medicine have opened up important landscapes for diagnosis and study of molecular mechanisms of congenital hemolytic anemias, genotyping has become a prerequisite for accessing new treatments and for evaluating disease state and progression. This review examines the extensive molecular heterogeneity of PK deficiency, focusing on the diagnostic impact of genotypes and new acquisitions on pathogenic non-canonical variants. The recent progress and the weakness in understanding the genotype-phenotype correlation, and its practical usefulness in light of new therapeutic opportunities for PK deficiency are also discussed.

Epidemiological Study of Hereditary Hemolytic Anemia in the Korean Pediatric Population during 1997-2016: a Nationwide Retrospective Cohort Study.

BACKGROUND: Hereditary hemolytic anemia (HHA) is a rare disease characterized by premature red blood cell (RBC) destruction due to intrinsic RBC defects. The RBC Disorder Working Party of the Korean Society of Hematology established and updated the standard operating procedure for making an accurate diagnosis of HHA since 2007. The aim of this study was to investigate a nationwide epidemiology of Korean HHA. METHODS: We collected the data of a newly diagnosed pediatric HHA cohort (2007-2016) and compared this cohort's characteristics with those of a previously surveyed pediatric HHA cohort (1997-2006) in Korea. Each participant's information was retrospectively collected by a questionnaire survey. RESULTS: A total of 369 children with HHA from 38 hospitals distributed in 16 of 17 districts of Korea were investigated. RBC membranopathies, hemoglobinopathies, RBC enzymopathies, and unknown etiologies accounted for 263 (71.3%), 59 (16.0%), 23 (6.2%), and 24 (6.5%) of the cases, respectively. Compared to the cohort from the previous decade, the proportions of hemoglobinopathies and RBC enzymopathies significantly increased (P < 0.001 and P = 0.008, respectively). Twenty-three of the 59 hemoglobinopathy patients had immigrant mothers, mostly from South-East Asia. CONCLUSION: In Korea, thalassemia traits have increased over the past 10 years, reflecting both increased awareness of this disease and increased international marriages. The enhanced recognition of RBC enzymopathies is due to advances in diagnostic technique; however, 6.5% of HHA patients still do not have a clear diagnosis. It is necessary to improve accessibility of diagnosing HHA.

How we manage patients with pyruvate kinase deficiency.

Novel therapies in development have brought a new focus on pyruvate kinase deficiency (PKD), the most common congenital haemolytic anaemia due to a glycolytic enzyme deficiency. With an improved recognition of its clinical presentation and understanding of the diagnostic pathway, more patients are likely to be identified with this anaemia. Complications, including gallstones and non-transfusion-related iron overload, require monitoring for early diagnosis and management. Current management remains supportive with red cell transfusions, chelation and splenectomy. Decisions to transfuse and/or splenectomise must be individualised. Haematopoietic stem cell transplant has been pursued in a small number of patients with mixed outcomes. Novel treatment approaches, which range from a small molecule pyruvate kinase activator to gene therapy, may transform the way in which PKD is managed in the future. In this review, we discuss the pathophysiology of PKD and present our approaches to diagnosis, monitoring and management of patients with this anaemia.

Addressing the diagnostic gaps in pyruvate kinase deficiency: Consensus recommendations on the diagnosis of pyruvate kinase deficiency.

Pyruvate kinase deficiency (PKD) is the most common enzyme defect of glycolysis and an important cause of hereditary, nonspherocytic hemolytic anemia. The disease has a worldwide geographical distribution but there are no verified data regarding its frequency. Difficulties in the diagnostic workflow and interpretation of PK enzyme assay likely play a role. By the creation of a global PKD International Working Group in 2016, involving 24 experts from 20 Centers of Expertise we studied the current gaps in the diagnosis of PKD in order to establish diagnostic guidelines. By means of a detailed survey and subsequent discussions, multiple aspects of the diagnosis of PKD were evaluated and discussed by members of Expert Centers from Europe, USA, and Asia directly involved in diagnosis. Broad consensus was reached among the Centers on many clinical and technical aspects of the diagnosis of PKD. The results of this study are here presented as recommendations for the diagnosis of PKD and used to prepare a diagnostic algorithm. This information might be helpful for other Centers to deliver timely and appropriate diagnosis and to increase awareness in PKD.

Appraisal of patient-reported outcome measures in analogous diseases and recommendations for use in phase II and III clinical trials of pyruvate kinase deficiency.

PURPOSE: Pyruvate kinase deficiency (PKD) is a rare disease and understanding of its epidemiology and associated burden remains limited. With no current curative therapy, clinical manifestations can be life threatening, clinically managed by maintaining adequate hemoglobin levels through transfusion and subsequent support, but with frequent complications. Treatment goals are to maintain/improve the patient's quality of life. With new therapies, reliable, valid, and relevant patient-reported outcome (PRO) tools are required for use in clinical trials. METHODS: Systematic literature search identified no current PRO tools for capturing/measuring the impact of PKD and treatments in clinical trials. Therefore, the search strategy was revised to consider conditions analogous to PKD in terms of symptoms and impacts that might serve as parallels to the experience in PKD; this included sickle cell anemia, thalassemia, and hemolytic anemia. Psychometric properties, strengths, and weakness of selected appropriate PRO instruments were compared, and recommendations made for choice of PRO tools. RESULTS: In adult populations, EORTC QLQ C30 and SF-36v2 are recommended, the former being a basic minimum, covering generic HRQoL, and core symptoms such as fatigue. In pediatric populations, PedsQL Generic Core Scale to measure HRQoL and PedsQL MFS scale to measure fatigue are recommended. CONCLUSIONS: Some symptoms/life impacts may be unique to PKD and not observable in analogous conditions. A 'Physico-Psychosocial Model' derived from the 'Medical Model' is proposed to form the basis for a hypothesized conceptual framework to address the development of PKD-specific PRO instruments.

The burden of disease in pyruvate kinase deficiency: Patients' perception of the impact on health-related quality of life.

OBJECTIVES: This study explored how signs and symptoms of pyruvate kinase (PK) deficiency, a rare hemolytic anemia caused by mutations in the PKLR gene, impacts patients' health-related quality of life (HRQoL). METHODS: Interviews with 21 adults with PK deficiency in the United States, Netherlands, and Germany were conducted. Participants were asked to describe signs, symptoms, and impacts of the disease on their daily lives. Interviews were transcribed and analyzed using qualitative analysis methods. RESULTS: The most common signs and symptoms reported were yellow eyes (n = 19), tiredness (n = 18), yellow skin (n = 17), fatigue (n = 15), low energy (n = 13), and shortness of breath (n = 13). Furthermore, signs and symptoms of PK deficiency negatively impact the ability to perform physical activities, appearance, social activities, emotional states, activities of daily living, leisure activities, work and/or school, sleep, and cognitive states of those living with PK deficiency. A conceptual model is presented that further demonstrates the profound impact that signs and symptoms of PK deficiency have on dimensions of patients' HRQoL. CONCLUSIONS: This is the first study that provides patient perspective on the burden of living with PK deficiency and lays the foundation for future studies to examine the effect of pharmacologic interventions on overall HRQoL for patients living with PK deficiency.

Targeted next generation sequencing for the diagnosis of patients with rare congenital anemias.

BACKGROUND: Most patients with anemia are diagnosed through clinical phenotype and basic laboratory testing. Nonetheless, in cases of rare congenital anemias, some patients remain undiagnosed despite undergoing an exhaustive workup. Genetic testing is complicated by the large number of genes involved in rare anemias and the similarities in the clinical presentation of the different syndromes. OBJECTIVE: We aimed to enhance the diagnosis of patients with congenital anemias by using targeted next-generation sequencing. METHODS: Genetic diagnosis was performed by gene capture followed by next-generation sequencing of 76 genes known to cause anemia syndromes. RESULTS: Genetic diagnosis was achieved in 13 out of 21 patients (62%). Six patients were diagnosed with pyruvate kinase deficiency, 4 with dehydrated hereditary stomatocytosis, 2 with sideroblastic anemia, and 1 with CDA type IV. Eight novel mutations were found. In 7 patients, the genetic diagnosis differed from the pretest presumed diagnosis. The mean lag time from presentation to diagnosis was over 13 years. CONCLUSIONS: Targeted next-generation sequencing led to an accurate diagnosis in over 60% of patients with rare anemias. These patients do not need further diagnostic workup. Earlier incorporation of this method into the workup of patients with congenital anemia may improve patients' care and enable genetic counseling.

A novel PKLR gene mutation identified using advanced molecular techniques.

This study's purposes were to diagnose intractable hemolytic anemia and to provide guiding treatment for the affected family members. We performed NGS in a panel of 600 genes for blood diseases on a patient with obscure hemolytic anemia and her parents. We confirmed the diagnosis of pyruvate kinase deficiency, identified a novel homozygous mutation of the PKLR gene (NM_000298: exon 6: c.T941C: p.I314T), and ruled out other blood diseases in the Chinese family. Furthermore, amniotic fluid was taken from the mother during the second trimester, and DNA was extracted to analyze the type of PKLR gene mutation. The proband received cord blood and bone marrow from the second child of the mother for hematopoietic stem cell transplantation and achieved normal hematopoiesis. The genetic characterization analysis and genotype-phenotype correlation study of PKLR gene suggested that NGS was an effective method to confirm the molecular diagnosis of intractable hemolytic anemia. The identification of the mutation aided in prenatal diagnosis in the second pregnancy and the effective clinical management of the affected family.

A Case With Pyruvate Kinase Deficiency Remarkably Sensitive to Heat.

Pyruvate kinase (PK) deficiency is the most common defect of the glycolytic pathway leading to congenital hemolytic anemia. We present the case of an 18-year-old boy with chronic nonspherocytic hemolytic anemia, who had remarkable sensitivity to heat. Moreover, the patient showed clinical impairment in the last year. For this reason, we excluded the immunologic or infectious nature (malaria, babesia), which may play a role in the worsening of anemia. Red blood cell enzyme assay showed the presence of a significant increase in other enzyme activities, except for PK, suggesting a PK deficiency in the patient. The molecular analysis of the PK-LR gene revealed the presence of a novel homozygote missense mutation (c.581G>C, p.Arg194Pro). The mutant enzyme displayed heat instability. In addition, we analyzed bilirubin uridine diphosphate (UDP)-glucuronosyltransferase 1A1 gene that revealed a heterozygous state ([TA]6/[TA]7). After a clear diagnosis of PK deficiency, the patient underwent splenectomy.

Cord blood transplantation in a young child with pyruvate kinase deficiency.

Unrelated cord blood transplantation (CBT) was performed for the treatment of pyruvate kinase (PK) deficiency in a female pediatric patient at the age of 1 year 7 months, who had been in severe and frequent transfusion-dependent hemolytic anemia, despite red blood cell (RBC) PK activity 5.52 IU/gHb. pyruvate kinase-liver and RBC (PK-LR) had a compound heterozygous mutation located on exon 8: c.1044G > T/c.1076G > A (K348N/R359H). Hemoglobin and RBC PK corrected to 13.5 g/dL and 9.00 IU/gHb, respectively, with gene correction at 6 months after CBT. CBT should be considered as an option for useful treatment in children with severe PK deficiency in the absence of HLA identical sibling with normal RBC PK activity.

Mortality among US veterans with a physician-documented diagnosis of pyruvate kinase deficiency.

Real-world studies of pyruvate kinase (PK) deficiency and estimates of mortality are lacking. This retrospective observational study aimed to identify patients with PK deficiency and compare their overall survival (OS) to that of a matched cohort without PK deficiency. Patients with ≥1 diagnosis code related to PK deficiency were selected from the US Veterans Health Administration (VHA) database (01/1995-07/2019); patients with a physician-documented diagnosis were included (PK deficiency cohort; index: date of first diagnosis code related to PK deficiency). Patients in the PK deficiency cohort were matched 1:5 to patients from the general VHA population (non-PK deficiency cohort; index: random visit date during match's index year). OS from index was compared between the two cohorts. Eighteen patients in the PK deficiency cohort were matched to 90 individuals in the non-PK deficiency cohort (both cohorts: mean age 57 years, 94% males; median follow-up 6.0 and 8.0 years, respectively). At follow-up, patients in the non-PK deficiency cohort had significantly longer OS than the PK deficiency cohort (median OS: 17.1 vs. 10.9 years; hazard ratio: 2.3; p = 0.0306). During their first-year post-index, 75% and 40% of the PK deficiency cohort had laboratory-confirmed anemia and iron overload, respectively. Among patients who died, cause of death was highly heterogeneous. These results highlight the increased risk of mortality and substantial clinical burden among patients with PK deficiency. While the intrinsic characteristics of the VHA database may limit the generalizability of the results, this is the first real-world study to characterize mortality in patients with PK deficiency.