Comorbidities and complications in adult and paediatric patients with pyruvate kinase deficiency: Analysis from the Peak Registry.

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.

Red cell distribution width and prognosis in myelofibrosis patients treated with ruxolitinib.

We evaluated RDW in a single-center series of 61 consecutive patients with primary and secondary MF at diagnosis and during treatment with ruxolitinib (RUX) and examined any possible prognostic impact. Elevated RDW values were present in all but 4 patients at diagnosis with a median RDW of 18.9%. RDW was higher in subjects with palpable splenomegaly (p = 0.02), higher ferritin, as well as among those cases who did not receive any cytoreduction before RUX (p = 0.04). Interestingly, higher RDW at diagnosis also correlated with a shorter time from MF diagnosis to RUX start (-4.1 months per one RDW unit; p = 0.03). We observed a modest increase (< 1%) in RDW during the first 6 months of RUX treatment. In a multivariable random-intercept model that considered all time points and contained the covariates time and RUX dose, we also observed a clear decrease in RDW with increasing hemoglobin (Hb) during RUX (slope: -0.4% per g/dL of Hb; p < 0.001). The median RDW at diagnosis of 18.9% was used as a cut-off to identify two subgroups of patients [Group 1: RDW 19.0-25.7%; Group 2: RDW 13.1-18.7%], showing a difference in mortality [Group 1 vs. 2: crude HR 2.88; p = 0.01]. Using continuous RDW at diagnosis, the crude HR was 1.21 per RDW unit (p = 0.002). In a Cox model adjusted for gender, age and Hb at diagnosis, the HR was 1.13 per RDW unit (p = 0.07). RDW may have prognostic significance at MF diagnosis and during RUX, helping in the rapid detection of patients with poor prognosis.

Glycolytic activity and in vitro effect of the pyruvate kinase activator AG-946 in red blood cells from low-risk myelodysplastic syndromes patients: A proof-of-concept study.

Glycolytic activity and in vitro effect of the pyruvate kinase activator AG-946 in red blood cells from low-risk myelodysplastic syndromes patients. Data showed decreased glycolytic activity in red blood cells of 2/3 of patients with lower-risk MDS. These results highlight a potential effect of the PK activator in this setting.

The impact of COVID-19 on menstrual cycle's alterations, in relation to depression and sleep disturbances: a prospective observational study in a population of medical students.

BACKGROUND: The sars-Cov-2 pandemic has determined psychological stress, particularly in the young population of medical students. We studied the impact of the pandemic on menstrual cycle alteration in relation to psychological stress, presence of depression, sleep disturbances and post-traumatic stress, on a population of medical students. METHODS: 293 female students at the Faculty of Medicine and Psychology of the Sapienza University of Rome (23.08 years old ± 3.8) were enrolled. In March 2021, one year after quarantine, a personal data sheet on menstrual cycle, examining the quality of the menstrual cycle during the pandemic, compared to the previous period. Concomitantly, the Beck Depression Inventory and the Impact of Event Scale have been administered. A Pearson chi-square test was assessed to evaluate the difference between the characteristics of the menstrual cycle and the scores obtained with the questionnaires. RESULTS: A statistically significant association between menstrual alterations and stress during pandemic had been found. The onset of depressive symptoms and sleep disturbances was observed in 57.1% and in 58.1% of young women with cycle's alterations, respectively. Amenorrhea was three times more common in female students with depressive symptoms, premenstrual syndrome had a significant correlation with both depression and sleep disturbances. The pandemic has been related to menstrual alterations, with depressive symptoms and sleep disorders. Amenorrhea is connected to depression, as observed on the functional hypothalamic amenorrhea. CONCLUSIONS: The pandemic affected the menstrual cycle as well as the depressive symptoms and sleep. Practical implications of the study lead to the development of strategies for psychological intervention during the pandemic experience, in order to help medical trainees, with specific attention to women's needs. Future studies should analyze the impact of other types of social stress events, on sleep, depression and the menstrual cycle beside the pandemic.

Changing trends of splenectomy in hereditary spherocytosis: The experience of a reference Centre in the last 40 years.

We describe the clinical/haematological characteristics of 446 patients with hereditary spherocytosis diagnosed in the last 40 years in a reference centre. The frequency of splenectomy decreased over time (44% before 1990 to 7% in 2011-2020), notwithstanding a confirmed good efficacy. Age at splenectomy progressively increased (63% in children before 1990 to 88% in patients aged ≥20 years in 2011-2020). Our real-life experience showed that even a fraction of patients in the trait/mild categories (19/92, 21%) were splenectomised, whilst 30/78 (38%) in the moderate/severe groups were not. Overall, these data pinpoint to the increasing awareness about post-splenectomy thromboses and infections.

Red blood cell phenotyping from 3D confocal images using artificial neural networks.

The investigation of cell shapes mostly relies on the manual classification of 2D images, causing a subjective and time consuming evaluation based on a portion of the cell surface. We present a dual-stage neural network architecture for analyzing fine shape details from confocal microscopy recordings in 3D. The system, tested on red blood cells, uses training data from both healthy donors and patients with a congenital blood disease, namely hereditary spherocytosis. Characteristic shape features are revealed from the spherical harmonics spectrum of each cell and are automatically processed to create a reproducible and unbiased shape recognition and classification. The results show the relation between the particular genetic mutation causing the disease and the shape profile. With the obtained 3D phenotypes, we suggest our method for diagnostics and theragnostics of blood diseases. Besides the application employed in this study, our algorithms can be easily adapted for the 3D shape phenotyping of other cell types and extend their use to other applications, such as industrial automated 3D quality control.

Recommendations for diagnosis and treatment of methemoglobinemia.

Methemoglobinemia is a rare disorder associated with oxidization of divalent ferro-iron of hemoglobin (Hb) to ferri-iron of methemoglobin (MetHb). Methemoglobinemia can result from either inherited or acquired processes. Acquired forms are the most common, mainly due to the exposure to substances that cause oxidation of the Hb both directly or indirectly. Inherited forms are due either to autosomal recessive variants in the CYB5R3 gene or to autosomal dominant variants in the globin genes, collectively known as HbM disease. Our recommendations are based on a systematic literature search. A series of questions regarding the key signs and symptoms, the methods for diagnosis, the clinical management in neonatal/childhood/adulthood period, and the therapeutic approach of methemoglobinemia were formulated and the relative recommendations were produced. An agreement was obtained using a Delphi-like approach and the experts panel reached a final consensus >75% of agreement for all the questions.

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.

The variable manifestations of disease in pyruvate kinase deficiency and their management.

Pyruvate kinase deficiency (PKD) is the most common cause of chronic hereditary non-spherocytic hemolytic anemia and results in a broad spectrum of disease. The diagnosis of PKD requires a high index of suspicion and judicious use of laboratory tests that may not always be informative, including pyruvate kinase enzyme assay and genetic analysis of the PKLR gene. A significant minority of patients with PKD have occult mutations in non-coding regions of PKLR which are missed on standard genetic tests. The biochemical consequences of PKD result in hemolytic anemia due to red cell pyruvate and ATP deficiency while simultaneously causing increased red cell 2,3-diphosphoglycerate, which facilitates oxygen unloading. This phenomenon, in addition to numerous other factors such as genetic background and differences in splenic function result in a poor correlation between symptoms and degree of anemia from patient to patient. Red cell transfusions should, therefore, be symptom-directed and not based on a hemoglobin threshold. Patients may experience specific complications, such as paravertebral extramedullary hematopoiesis and chronic debilitating icterus, which require personalized treatment. The decision to perform splenectomy or hematopoietic stem cell transplantation is nuanced and depends on disease burden and long-term outlook given that targeted therapeutics are in development. In recognition of the complicated nature of the disease and its management and the limitations of the PKD literature, an international working group of ten PKD experts convened to better define the disease burden and manifestations. This article summarizes the conclusions of this working group and is a guide for clinicians and investigators caring for patients with PKD.

Genotype-phenotype correlation and molecular heterogeneity in pyruvate kinase deficiency.

Pyruvate kinase (PK) deficiency is a rare recessive congenital hemolytic anemia caused by mutations in the PKLR gene. This study reports the molecular features of 257 patients enrolled in the PKD Natural History Study. Of the 127 different pathogenic variants detected, 84 were missense and 43 non-missense, including 20 stop-gain, 11 affecting splicing, five large deletions, four in-frame indels, and three promoter variants. Within the 177 unrelated patients, 35 were homozygous and 142 compound heterozygous (77 for two missense, 48 for one missense and one non-missense, and 17 for two non-missense variants); the two most frequent mutations were p.R510Q in 23% and p.R486W in 9% of mutated alleles. Fifty-five (21%) patients were found to have at least one previously unreported variant with 45 newly described mutations. Patients with two non-missense mutations had lower hemoglobin levels, higher numbers of lifetime transfusions, and higher rates of complications including iron overload, extramedullary hematopoiesis, and pulmonary hypertension. Rare severe complications, including lower extremity ulcerations and hepatic failure, were seen more frequently in patients with non-missense mutations or with missense mutations characterized by severe protein instability. The PKLR genotype did not correlate with the frequency of complications in utero or in the newborn period. With ICCs ranging from 0.4 to 0.61, about the same degree of clinical similarity exists within siblings as it does between siblings, in terms of hemoglobin, total bilirubin, splenectomy status, and cholecystectomy status. Pregnancy outcomes were similar across genotypes in PK deficient women. This report confirms the wide genetic heterogeneity of PK deficiency.

Iron overload in congenital haemolytic anaemias: role of hepcidin and cytokines and predictive value of ferritin and transferrin saturation.

Iron overload (IO) is poorly investigated in the congenital haemolytic anaemias (CHAs), a heterogeneous group of rare inherited diseases encompassing abnormalities of the erythrocyte membrane and metabolism, and defects of the erythropoiesis. In this study we systematically evaluated routine iron parameters and cardiac and hepatic magnetic resonance imaging, together with erythropoietin, hepcidin, non-transferrin bound iron (NTBI), and cytokine serum levels in patients with different CHAs. We found that 40% of patients had a liver iron concentration (LIC) >4 mg Fe/g dry weight. Hepatic IO was associated with ferritin levels (P = 0·0025), transferrin saturation (TfSat, P = 0·002) and NTBI (P = 0·003). Moreover, ferritin >500 µg/l plus TfSat >60% was demonstrated as the best combination able to identify increased LIC, and TfSat alteration as more important in cases with discordant values. Possible confounding factors, such as transfusions, hepatic disease, metabolic syndrome and hereditary haemochromatosis-associated mutations, had negligible effects on IO. Erythropoietin and hepcidin levels were increased in CHAs compared with controls, correlating with LIC and ferritin, respectively. Regarding cytokines, γ-interferon (IFN-γ) was increased, and both interleukin 6 and IFN-γ levels positively correlated with ferritin and hepcidin levels. Overall, these findings suggest the existence of a vicious cycle between chronic haemolysis, inflammatory response and IO in CHAs.

Integrating clinical, morphological, and molecular data to assess prognosis in patients with primary myelofibrosis at diagnosis: A practical approach.

Currently available prognostic scoring systems in primary myelofibrosis (PMF) do not integrate clinical, histological, and molecular data, or they also required information on "other" mutations that are available in the clinical practice only in a very limited number of laboratories. In the present multicenter study, including 401 PMF patients, an integrated International Prognostic Scoring System (I-IPSS) was developed by combining IPSS, grade of bone marrow fibrosis (GBMF), and driver mutations molecular status (MS) to define PMF prognosis at diagnosis. Four prognostic categories were identified: I-IPSS-low risk (113 patients), I-IPSS-intermediate-1 risk (56 patients), I-IPSS-intermediate-2 risk (154 patients), and I-IPSS-high risk (78 patients). Median overall survival was 26.7 years in I-IPSS-intermediate-1, 10.8 in I-IPSS-intermediate-2, and 6.4 in I-IPSS-high-risk patients (log-rank test <0.0001); instead, it was not reached in the I-IPSS-low-risk cohort because of the extremely low number of registered deaths. The addition of GBMF and MS to IPSS improved the efficacy for predicting the risk of death. Indeed, the sensitivity of I-IPSS was significantly higher (P < .05) than that of IPSS, considering both total deaths and 5- and 10-year mortality. This comprehensive approach allows clinicians to evaluate mutual interactions between IPSS, GBMF, and MS and identify high-risk patients with poor prognosis who may benefit from aggressive treatments. More importantly, this integrated score can be easily applicable worldwide as it only required information that represent the good clinical practice in the management of PMF patients.

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.

A New Variant of PKLR Gene Associated With Mild Hemolysis may be Responsible for the Misdiagnosis in Pyruvate Kinase Deficiency.

Pyruvate kinase deficiency (PKD) is the most common glycolytic defect leading to hemolytic anemia. PKD is caused by the mutations in the PKLR gene; however, the detection of a decreased PK activity should be first measured for rapid diagnosis. We report here the case of a 1-year-old girl with mild hemolysis and PKD. At the time of the study, the patient showed a hemoglobin level of 9.5 g/dL, mean corpuscular volume of 93 fL, reticulocyte of 6.7%, and lactate dehydrogenase of 218 IU/L. Peripheral blood smear showed polychromasia, anisocytosis, tear drop cells, fragmented eyrtrocytes, and target cells. When a biochemical analysis was performed in our patient and her parents who had consanguinity, a decreased PK activity was detected in the patient and her father. After the molecular study of PKLR gene, a new homozygote variant, c.1708G>T (pVal570Leu), was found in our patient and her father. Her father had a misdiagnosis of Gilbert syndrome because he had unconjugated hyperbilirubinemia and not anemia. Her mother was also a carrier of the mutation in heterozygous state. Patients presenting with hemolytic anemia, either severe or mild hemolytic anemia, should be screened for PKD in the first year of life. Patients with mild hemolytic findings can be followed-up with misdiagnoses.

A New Homozygous Mutation (c.393-394del TA/c.393-394del TA) in the NT5C3 Gene Associated With Pyrimidine-5'-Nucleotidase Deficiency: A Case Report.

Pyrimidine-5-nucleotidase (P5'N-1) deficiency is a rare nonspherocytic hemolytic anemia due to pyrimidine nucleotide deposition within erythrocytes. This rare erythrocyte disorder shows autosomal recessive inheritance with mutation of the pyrimidine-5'-nucleotidase gene, which is localized on 7p15-p14. Consanguinity of parents increases the probability of disease with novel mutations. Here, we report a 12-year-old boy with a delayed diagnosis of P5'N deficiency whose parents were consanguineous. He had a hemoglobin level of 7.5 g/dL, mean corpuscular volume of 93 fL, 7% reticulocyte, and lactate dehydrogenase of 678 IU/L. A peripheral blood smear showed polychromasia, marked anisopoikilocytosis with schistocytes, elliptocytes, stomatocytes, spherocytes, dacryocyte, and basophilic stippling in red blood. Decreased purine/pyrimidine ratio was 1.07 (normal range=1.4 to 2.98). Molecular analysis with direct DNA sequencing of the NT5C3 gene, codifying for P5'N-1, revealed the presence of a novel homozygous mutation, c393-394delTA, in the gene coding P5'N enzyme in the patient. To our knowledge, this is a newly defined mutation in P5'N deficiency.

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.