Cases Of Pyruvate Kinase Deficiency Research Articles

CLINICOETIOLOGICAL STUDY OF HEMOLYTIC ANAEMIA IN CHILDREN PRESENTING TO A TERTIARY CARE HOSPITAL OFKASHMIR

Anemia is said to be present when the hemoglobin levels are more than two standard deviations below the mean for the childs age and sex. Hemolytic anemias arise when red blood cells have decreased survival either due to an intrinsic abnormality of the cell or due to extrinsic factors or both. Hemolytic anemias constitute important cause of mortality and morbidity in developing countries. These group of anemias have various clinical presentations starting from their age of onset of symptoms, failure to thrive, anemia, jaundice, splenomegaly, cholelithiasis, cardiomegaly, congestive cardiac failure, severe life-threatening infections and chronic disabilities leading to distress in the families. The present study attempts to reveal the clinical and etiological profile of patients with different types of hemolytic anemia admitted in a pediatric tertiary care hospital of Kashmir. Review of literature shows only few epidemiological studies particularly in this part of the country, and no such study in our state though the disease is fairly prevalent. We undertook this study with this idea. Aims and Objectives: The aims and objectives of this study was to determine clinical and etiological profile of hemolytic anemia in children aged 1month to 18 years admitted in tertiary care hospital of Kashmir. Materiaals & Methods: This prospective observational study was carried out in a pediatric tertiary care hospital in Kashmir over a period of two years. Prior to initiation of study, ethical clearance was obtained. All patients admitted in the indoor of department of pediatrics between age group of 1 month to 18 years with evidence of hemolysis were included in the study. 50 cases were included in the study after obtaining written informed consent from parent or guardian. A detailed history, examination and required investigations were collected. Data was entered and assessed in MS Excel. Results: The study was based on 50 patients, almost equal males and females, 1month to 18 years of age. The most common hemolytic anemia was beta thalassemia major (n=17, 34%), followed by autoimmune hemolytic anemia(n=10,20%), hereditary spherocytosis (n=5,10%), thalassemia trait (8%), pyruvate kinase deficiency (8%) and glucose 6 phosphate dehydrogenase (8%), thalassemia intermedia (4%), sickle cell trait (2%), microangiopathic hemolytic anemia (2%), hereditary xerosis (2%) and hereditary stomatocytosis (2%). Highest number of the cases was less than 3 years old (56%). The most common clinical presentation was jaundice, which was present in all cases, followed by splenomegaly (60%), hepatomegaly (30%), hemolytic facies (26%). Splenomegaly was seen in all cases of thalassemia major, thalassemia intermedia, beta thalassemia trait, hereditary spherocytosis, 2 cases of pyruvate kinase deficiency and 3 cases of glucose 6 phosphate dehydrogenase deficiency. Spleen >5cm was seen in 10 patients. Massive splenomegaly with abdominal discomfort was seen in 3 cases. The mean hemoglobin was least in beta thalassemia major followed by beta thalassemia intermedia, Hereditary spherocytosis, autoimmune hemolytic anemia, glucose 6 phosphate dehydrogenase deficiency and the highest mean hemoglobin was seen in pyruvate kinase deficiency. Means for serum ferritin and serum bilirubin were calculated.

Use of Next Generation Sequencing Panel to Clarify Undiagnosed Cases of Hereditary Hemolytic Anemias

Background. Hereditary chronic hemolytic anemias (CHAs) is an heterogeneous group of rare disorders including defects of the RBC membrane (hereditary spherocytosis (HS), elliptocytosis (HE), pyropoikilocytosis (HPP), or defects of cation permeability, hereditary stomatocytosis (HSt)), defects of RBC metabolism (affecting glycolysis, the pentose-phosphate shunt or nucleotide metabolism), hemoglobinopathies, and disorders of erythropoiesis (e.g. congenital dyserythropoietic anemia, CDAs). All these diseases are complex and frequently difficult to diagnose. The laboratory tools for the diagnosis of CHAs include routine tests but also require more specialized analyses, available only in few dedicated laboratories. In our experience, a conclusive diagnosis is not reached in about 15-20% of cases despite detailed and exhaustive investigations. Genetic analysis is not widely used in these disorders due to the clinical and molecular heterogeneity, and is usually performed only when the hematologic diagnostic workup gives a clear diagnostic orientation. The advent of next generation sequencing (NGS) methods however has made diagnosing complex genetic disorders feasible.Aim. We developed and applied a NGS targeted panel to identify the underlying genetic cause in a selected series of patients with undiagnosed CHAs.Methods. A total of 35 cases with CHAs from 26 families were studied. In 21 subjects no definitive diagnosis was previously done despite extensive investigation, in 7 the conventional diagnostic workup was unreliable due to transfusions or sample shipping. In addition three HS/HE families (7 cases) with intra-family clinical variability were studied to elucidate the molecular basis of the atypical phenotype. Using SureDesign software (Agilent) we created a NGS based panel containing 40 genes associated with RBC membrane disorders (13 genes), enzymopathies (20), CDAs (7), and 9 other candidate or modifier genes. Libraries were obtained by HaloPlexHS Target Enrichment System Kit and sequenced on a MiSeq platform (Illumina). The panel was validated on previously characterized CHAs patients. Mutations were confirmed by Sanger method.Results. We identified pathogenic variants that enabled a definitive diagnosis in 19 cases (13 families). Results are reported in Table 1.RBC enzyme defects were identified in 5 patients: two cases of pyruvate kinase deficiency (one of them with normal PK activity in absence of transfusions), one case of glucosephosphate isomerase deficiency, and one of NADH-CytB5R deficiency (in a case with methemoglobinemia). Notably, we were able to detect a triosephosphate isomerase deficiency in a 7 month child with anemia but no neuromuscular manifestations at the time of the study, thus permitting an early diagnosis in this rare disease.Dyserythropoietic anemias. SEC23B gene mutations associated with CDAII were identified in one patient. A new pathogenic variant in ALAS2 gene was detected in a female with macrocytic anemia, suggesting a diagnosis of congenital sideroblastic anemia; HUMARA analysis showed a skewed X-Chr inactivation pattern; the mutation was transmitted by the asymptomatic mother.RBC membrane defects. Three cases carried the known pathogenic variant p.R2456H in PIEZO1 gene, associated with hereditary xerocytosis. A mutation in ABCG8 gene associated with congenital sitosterolemia was detected in case F10-413, who had a long-lasting diagnosis of overhydrated HSt suggested by a right shifted ekacytometric curve.Complex genotypes were identified in 3 families with HS/HE: case F11-817 (mixed HS/HE phenotype) carried two pathogenic variants in SPTA1 and SLC4A1, whereas the brother with typical HE had only the SPTA1 variant; case F12-73 with HPP and combined 68% spectrin and 56% ankyrin deficiency, had two different in trans mutations in SPTA1, one of them associated in cis with the alpha-LELY allele. Finally, case F13-723 (severe HS with 50% band 3 deficiency) had 3 different variants in SLC4A1 gene, the father presenting only one missense mutation, thus justifying intra-family clinical variability.Conclusions. NGS platform is a powerful tool to elucidate the molecular causes in patients with CHAs where traditional hematologic testing failed, leading in this series to the clarification of 50% of the examined families. Extensive clinical and hematologic information is needed to support the interpretation of molecular data. [Display omitted] Disclosuresvan Wijk:Agios Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Barcellini:Alexion: Honoraria; Agios: Honoraria, Research Funding; Novartis: Honoraria. Bianchi:Agios Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees.

Diagnostic challenges in pyruvate kinase deficiency

Red cell pyruvate kinase deficiency is a rare congenital, nonspherocytic hemolytic anemia caused by a glycolytic defect that is due to compound heterozygous or homozygous mutations in the PKLR gene on chromosome 1q21. The article presents analytical review of literature and the clinical case of pyruvate kinase deficiency. Patient's parents agreed to use personal dats and photos in research and publications.

Genetic diagnosis of erythrocyte pyruvate kinase deficiency and discovery of PKLR gene novel mutations

Objective To investigate the clinical symptoms of pyruvate kinase deficiency (PKD) and the new mutation type of PKLR gene in 3 cases of PKD, and to explore the method for PKD gene diagnosis. Methods Sequencing of blood system-related genes in 3 children was performed by target sequence capture and high-throughput sequencing technology, and the protein function of mutant gene was forecasted, after detecting the pathogenicity of the patients, these genotypes were confirmed by Sanger sequencing. Results In the 3 children, 5 types of PKLR gene mutations were found: double heterozygous mutations c. 1529G>A(p.R510Q)and c. 1031T>G(p.I344S), homozygous mutation c. 847G> T (p.V283F), double heterozygous mutations c. 979delC(p.L327fs)and c. 604_617del(p.V202fs). PKLR gene c. 1529G>A(p.R510Q)mutation had been reported previously, and the other four mutations were new.c.1031T> G (p.I344S) and c. 847G> T (p.V283F) was possibly pathogenic mutation, which meant that the probability of mutation of this gene was more than 90% and c. 979delC (p.L327fs) and c. 604_617del (p.V202fs) variation was a pathogenic variation.These 5 mutations had a greater effect on protein function, and all of them were pathogenic mutations. Conclusion Since PKD patients are difficult to be diagnosed clinically, PKLR gene variation can be detected by target sequence capture and high throughput sequencing technology, and the pathogenicity of the new mutant is evaluated. Key words: Pyruvate kinase; Mutation; PKLR gene

Rescue of Pyruvate Kinase Deficiency in Mice by Gene Therapy Using the Human Isoenzyme

Human erythrocyte R-type pyruvate kinase deficiency (PKD) is a disorder caused by mutations in the PKLR gene that produces chronic nonspherocytic hemolytic anemia. Besides periodic blood transfusion and splenectomy, severe cases require bone marrow (BM) transplant, which makes this disease a good candidate for gene therapy. Here, the normal human R-type pyruvate kinase (hRPK) complementary (cDNA) was expressed in hematopoietic stem cells (HSCs) derived from pklr deficient mice, using a retroviral vector system. These mice show a similar red blood cell phenotype to that observed in human PKD. Transduced HSCs were transplanted into myeloablated adult PKD mice or in utero injected into nonconditioned PKD fetuses. In the myeloablated recipients, the hematological manifestations of PKD were completely resolved and normal percentages of late erythroid progenitors, reticulocyte and erythrocyte counts, hemoglobin levels and erythrocyte biochemistry were restored. Corrected cells preserved their rescuing capacity after secondary and tertiary transplant. When corrected cells were in utero transplanted, partial correction of the erythrocyte disease was obtained, although a very low number of corrected cells became engrafted, suggesting a different efficiency of cell therapy applied in utero. Our data suggest that transduction of human RPK cDNA in PKLR mutated HSCs could be an effective strategy in severe cases of PKD.

Pyruvate kinase deficient hemolytic anemia in the Northern Irish population

A common cause of hereditary nonspherocytic hemolytic anemia is pyruvate kinase deficiency, which is associated with lifelong chronic hemolysis. Pyruvate kinase deficiency has a worldwide distribution with a higher prevalence in the Caucasian population, and especially in Europe and North America. It is inherited in an autosomal fashion and over 180 different mutations have been described. Investigation of hemolytic anemia in Northern Ireland has uncovered 4 new cases of pyruvate kinase deficiency. Molecular investigation revealed a total of six different mutations. One mutation (p.Arg495Val) is reported here for the first time in a homozygous patient. Another mutant PKLR allele harbored a nonsense and frameshift mutation in cis: c.[721G > T; 826delG]. Considering the three previously described Irish cases of pyruvate kinase deficiency, this study raises the total number of pyruvate kinase-deficient Irish patients to seven in which a total of nine mutant PKLR alleles were identified. This indicates the absence of a founder pyruvate kinase mutation in the Northern Ireland population. Although pyruvate kinase deficiency is prevalent in the Caucasian population it is not reflected in the number of individuals diagnosed in Northern Ireland. Hence, many cases of pyruvate kinase deficiency may remain undetected possibly due to the resultant anemia being mild.

Failure of partial splenectomy to ameliorate the anemia of pyruvate kinase deficiency

The authors report a case of pyruvate kinase deficiency in a 4-year-old patient who required monthly blood transfusions. A partial splenectomy was performed to decrease the transfusion requirements and at the same time preserve splenic function. After removal of 80% of splenic tissue there was no amelioration of her transfusion needs. She responded well to subsequent total splenectomy performed 6 months later, and is currently well and transfusion independent. Rapid splenic regeneration possibly caused by erythrophagocytosis and extramedullary hematopoiesis is the presumed reason for this patient's failure to respond to partial splenectomy.

An Autopsy Case of Pyruvate Kinase Deficiency AnemiaAssociated with Severe Hemochromatosis.

We report an autopsy case of pyruvate kinase deficiency anemia with severe hemochromatosis. This anemia is rarely associated with hemochromatosis. In this case, the autopsy findings showed hemochromatosis of the heart, pancreas, liver, kidneys, thyroid gland, adrenal glands, testes and skin. Microscopic examination showed iron depositions in these organs, but not in the bone marrow. A family study showed negative data for iron overload and no known HLA type suggestive of idiopathic hemochromatosis. To explain this rare association, we suggest that this patient's iron overload was an acquired type, which might have mainly been caused by increased iron absorption due to the severe hemolytic anemia.