Red Cell Pyruvate Kinase Deficiency Research Articles

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 >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.

A Family Affected by a Life-Threatening Erythrocyte Defect Caused by Pyruvate Kinase Deficiency With Normal Iron Status: A Case Report.

BackgroundRed cell pyruvate kinase deficiency (PKD) is a defect of glycolysis causing congenital non-spherocytic hemolytic anemia. PKD is transmitted as an autosomal recessive trait. The clinical features of PKD are highly variable, from mild to life-threatening anemia which can lead to death in the neonatal period. Most patients with PKD must receive regular transfusions in early childhood and as a consequence suffer from iron overloading.PatientHere, we report a Polish family with life-threatening hemolytic anemia of unknown etiology. Whole exome sequencing identified two heterozygous mutations, c.1529 G > A (p.R510Q) and c.1495 T > C (p.S499P) in the PKLR gene. Molecular modeling showed that the both PKLR mutations are responsible for major disturbance of the protein structure and functioning. Despite frequent transfusions the patients do not show any signs of iron overload and hepcidin, a major regulator of iron uptake, is undetectable in their serum. The patients were homozygous for the rs855791 variant of the TMPRSS6 gene which has earlier been shown to down-regulate iron absorption and accumulation.ConclusionThe lack of iron overload despite a reduced level of hepcidin in two transfusion-dependent PKD patients suggests the existence of a hepcidin-independent mechanism of iron regulation preventing iron overloading.

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.

Pyruvate kinase deficiency: epidemiology, molecular analyses and modern diagnostic approaches (literature review)

Red cell pyruvate kinase deficiency is the most common glycolytic defect causing congenital nonspherocytic hemolytic anemia. Pyruvate kinase is the enzyme involved in the last step of glycolysis – the transfer of a phosphate group from phosphoenolpyruvate producing the enolate of pyruvate and ATP (50 % of total energy ATP of erythrocytes). ATP deficiency directly shortened red cell lifespan. Affected red blood cells are destroyed in the splenic capillaries, leading to the development of chronic hemolytic anemia. It is an autosomal recessive disease, caused by homozygous and compound heterozygous mutations in the PKLR gene. There are no exact data on the incidence of pyruvate kinase deficiency, but the estimated frequency varies from 3: 1,000,000 to 1:20,000. The clinical features of the disease and the severity are highly variable. Diagnosis of pyruvate kinase deficiency is based on the determination of pyruvate kinase activity and molecular genetic study of the PKLR gene. The variety of clinical manifestations, possible complications, as well as the inaccessibility of diagnostic methods complicate the diagnosis.

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.

Prevalence of Red Cell Pyruvate Kinase Deficiency: A Systematic Literature Review

Introduction: Red cell pyruvate kinase deficiency (PKD) is a rare congenital disorder caused by compound heterozygosity or homozygosity for >300 mutations in the PKLR gene. The resulting glycolytic defect can lead to lifelong chronic hemolysis and associated symptoms, including anemia, jaundice, and iron overload. Reports of PKD prevalence vary, likely due to several factors, including PKD rarity, differences in measurement approaches, diagnostic challenges, and variable clinical expression. To understand the prevalence of PKD, we conducted a systematic literature review to identify and evaluate analyses of PKD epidemiology or PKLR mutant allele frequency (MAF). Methods: We queried Embase and Medline, screened conference abstracts, and considered relevant articles encountered incidentally to identify peer-reviewed references published before January 23, 2019 reporting PKD prevalence or incidence, PKLR MAF among the general population, or crude results from which prevalence, incidence or MAF could be derived. Two independent reviewers screened for eligibility and extracted data from eligible studies using a custom tool. Results: Of 1390 references screened, 1296 were excluded after title/abstract review (Figure). Of the remaining 94 references, 34 met eligibility criteria on full text review. In 30/34 eligible studies, potential sources of bias in PKD prevalence were identified, such as a non-generalizable study population (e.g., malaria-endemic areas; n=17), use of diagnostic assays of questionable accuracy (n=9), consideration of mutations with incomplete penetrance (n=3), and consideration of mutations with unclear clinical significance (n=1). The remaining 4 studies were considered high-quality for the estimation of PKD prevalence and were further assessed. Among these 4 studies, an important distinction was made between studies reporting diagnosed prevalence (n=3) and overall disease prevalence (diagnosed and undiagnosed PKD; n=1). Two studies estimated diagnosed PKD prevalence as 3.2 per million[i] and 8.5 per million[ii] by identifying diagnosed PKD cases from source populations of known size. We estimated the prevalence of diagnosed PKD in a general population to be 6.5 per million using data from another high-quality study[iii] that screened newborns for bilirubin and tested jaundiced newborns for PKD. These 3 studies are likely underestimates because they only consider diagnosed cases, and in one study only diagnosed, jaundice cases. In the final study,[iv] the authors sought to limit biases related to the Hardy-Weinberg equilibrium (HWE) assumption, as HWE incorrectly assumes the penetrance of each mutation is 100%. To address this, the authors identified a mutation known to have high penetrance (c.1529G>A), referred to as the 'index mutation'. They then assumed that the frequency of the index mutation relative to other PKD-causing mutations is the same between the general population and PKD cases, which led to a prevalence estimate (diagnosed and undiagnosed) of 51 per million (standard error: 32.5 per million). Conclusions: We consider the likely range of diagnosed PKD prevalence in general Western populations to be 3.2 to 8.5 per million. However, diagnosed and undiagnosed PKD prevalence may be as high as 51 per million. Future studies are needed to understand the clinical significance of various mutant alleles, which may inform more accurate, clinically-relevant PKD prevalence estimates, identify the degree of and reasons for underdiagnosis, and elucidate PKD heterogeneity between populations.

Bone Marrow Transplantation As A Curative Treatment In Red Cell Pyruvate Kinase Deficiency

Bone Marrow Transplantation As A Curative Treatment In Red Cell Pyruvate Kinase Deficiency

Bone marrow transplantation as permanent curative treatment in red cell pyruvate kinase deficiency

Bone marrow transplantation as permanent curative treatment in red cell pyruvate kinase deficiency

Alu-Element Insertion in Pklr Gene As a Novel Cause of Severe Hereditary Nonspherocytic Hemolytic Anemia

Red blood cell pyruvate kinase deficiency (PKD) is the most frequent enzyme abnormality of the glycolytic pathway and the most common cause of hereditary nonspherocytic hemolytic anemia. Its incidence has been estimated to be 1 in 20,000, especially in regions where consanguinity is common. It is inherited in autosomal recessive manner occurring as result of homozygous or compound heterozygous mutations affecting both alleles in PKLR gene. Over 200 mutations have been described in patients with PKD causing various severity of hemolysis. Most cases are due to missense mutations, but small deletions, insertions, splice site alterations, frame shifts, disruption of erythroid specific promoters, and nonsense mutations have been reported.Alu elements are the most abundant mobile DNA sequences in the human genome, contributing to almost 11% of its mass. These mobile elements have contributed significantly to evolution and human diversity by causing gene rearrangements. Alu insertions particularly have been associated with a number of human diseases either by disrupting a coding region or a splice signal. In this abstract, we report an Alu-element insertion in the coding region of PKLR gene as a novel cause of PKD causing severe hereditary nonspherocytic hemolytic anemia.The proband was a 16-month-old middle eastern male born to consanguineous parents. He was diagnosed with severe chronic hemolytic anemia and neonatal jaundice requiring regular blood transfusion since birth. There were no known family members with anemia requiring transfusions. The patient's RBC phenotypic analysis was challenging since most of the circulating RBCs were donor cells. Using a Next-Generation sequencing panel for 27 hemolytic anemia associated genes, we identified and characterized a novel homozygous insertional mutation in exon 6 of PKLR gene along with heterozygosity for ANK1 c.1404+15C>T variant. The mutation in exon 6 was identified as Alu element insertion. The inserted Alu element (~370 bp in size), belonging to the youngest Yb8 subfamily, disrupts the reading frame at isoleucine 314 in exon 6 of PKLR gene, leading to a premature stop codon within the inserted sequence. Quantitative reverse transcription-PCR (qRT-PCR) in the patient's reticulocyte RNA using primers for the sequence before the Alu insertion revealed a transcript present but decreased by 80% compared to the average of controls' reticulocyte RNA. This truncated transcript even if translated is expected to cause severe enzyme deficiency.Both parents had a normal hemoglobin level with mild reticulocytosis. The mother had a PK enzyme activity of 4.5 units/g Hb (normal range 6.7-14.3 units/g Hb) indicating that she is heterozygous for PKD. Paternal PK activity level was not determined due to insufficient sample. Carrier testing for mutation in PKLR gene in both parents is currently in process at the time of this submission.The variant in ANK1 gene was predicted to be damaging by affecting the splice site in ankyrin gene. However the father was also heterozygous for this variant and his ektacytometry was normal suggesting a benign nature of ANK1 c.1404+15C>T.This case represents, to our knowledge, the first report of a pathogenic PKLR mutation due to an Alu element insertion and demonstrates a novel mechanism causing severe hereditary nonspherocytic hemolytic anemia. This report also illustrates the challenge of diagnosing congenital hemolytic anemias in chronically transfused patients. The patients often present with a complex clinical picture and ambiguous laboratory findings and may have several potentially damaging genetics variants identified by next generation sequencing. A critical evaluation of the clinical symptoms, laboratory findings and genetic data from other family members and bioinformatics analysis are required to clarify the contribution of these variants and to arrive at an accurate diagnosis, which will guide the institution of targeted interventions and appropriate genetic counseling. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Erythrocyte pyruvate kinase deficiency: 2015 status report.

Over the last several decades, our understanding of the genetic variation, pathophysiology, and complications of the hemolytic anemia associated with red cell pyruvate kinase deficiency (PKD) has expanded. Nonetheless, there remain significant gaps in our knowledge with regard to clinical care and monitoring. Treatment remains supportive with phototherapy and/or exchange transfusion in the newborn period, regular or intermittent red cell transfusions in children and adults, and splenectomy to decrease transfusion requirements and/or anemia related symptoms. In this article, we review the clinical diversity of PKD, the current standard of treatment and for supportive care, the complications observed, and future treatment directions.Am. J. Hematol. 90:825–830, 2015. © 2015 Wiley Periodicals, Inc.

A Case of Congenital Red Cell Pyruvate Kinase Deficiency Associated with Hereditary Spherocytosis

Abstract 5272Pyruvate kinase (PK) deficiency, transmitted as an autosomal recessive trait, is the most common erythroenzymopathy of glycolytic pathway (prevalence of 1:20,000) associated with chronic non spherocytic hemolytic anemia from mild to severe. More than 180 mutations in the PK-LR gene have been so far reported, and genotype-phenotype correlation has been established for some of them.Hereditary Spherocytosis (HS) is the most common congenital hemolytic anemia in Caucasians, with an estimated prevalence ranging from 1:2000 to 1:5000. The main clinical features are hemolytic anemia from compensated to severe, variable jaundice, splenomegaly and cholelythiasis. The molecular defect is highly heterogeneous, caused by proteins involved in the attachment of cytoskeleton to the membrane integral domain (spectrin, ankyrin, band 3 and protein 4.2).We describe a case of PK deficiency associated with HS. The propositus was a 13 years-old Italian male with neonatal jaundice and need of blood transfusion (Hb 5.8 g/dL) during an infectious episode. At the time of the study Hb was 13.9 g/dL, MCV 81.8 fL, reticulocytes 207×109/L, unconjugated bilirubin 2.16 mg/dL, LDH 605 U/L, haptoglobin <20 mg/dL. The peripheral blood smear examination showed the presence of spherocytes (16%) and some ovalocytes (2%). The study of the most important red cell enzymes revealed reduced PK activity (59% of normal). Direct sequencing of PK-LR gene showed compound heterozygosity for the 994A mutation (Gly332Ser) and the −148T variant localized the erythroid specific promoter region.The presence of spherocytes in peripheral blood smear prompted us to investigate for the coexistence of HS. Erythrocyte osmotic fragility was decreased and SDS–PAGE analysis of red cell membrane proteins revealed a 30% spectrin reduction.Family study demonstrated a heterozygous condition for the 994A mutation in the father, who also displayed comparable enzyme deficiency, whereas promoter variant −148T was detected in the mother and in the brother. No red cell membrane abnormalities were present in the family members, although positive EMA binding test and increased osmotic fragility were found in the father and brother. The co-existence of HS and PK deficiency is very rare event, only few cases are described to date. Clinical, family and molecular studies allowed the determination of the interrelationship between the two RBC abnormalities in the patient and his relatives. The reduced PK activity in the propositus and his father is justified by heterozygous 994A mutation. The more severe clinical picture in the propositus could be caused by the coexistence of HS and by the presence of −148T mutation, that although it seems not to have effects on PK-LR mRNA expression, is often detected in PK deficient subjects with heterozygous PK mutations. Disclosures:No relevant conflicts of interest to declare.

Red cell pyruvate kinase deficiency in Southern Sardinia

Pyruvate kinase (PK) deficiency is the most frequent red cell enzymatic defect responsible for hereditary non-spherocytic hemolytic anemia. The clinical picture is quite variable and the reasons of this variability have been only partially clarified. We report the clinical description and the extended molecular analysis in 3 PK deficient patients with clinical phenotype of variable severity. We studied the clinical and hematological aspects of 3 patients and analyzed the following genes: pyruvate kinase-R, glucose-6-phosphate-dehydrogenase, α-globin, uridindiphosphoglucuronil transferase and HFE. One patient (A) with a severe clinical picture resulted homozygote for exon 8 nt994A substitution, the other 2 (brothers) were compound heterozygotes for exon 8 nt994A and exon 11 nt1456T mutation. One of the two brothers with a more severe phenotype coinherited also had G6PD deficiency, while both had microcytosis due to the homozygosity for the non-deletional form of α-thalassemia ATG → ACG substitution at the initiation codon of the alpha2 globin gene. Our results suggest that extended molecular analysis is useful for studying how several interacting gene mutations contribute to the clinical variability of pyruvate kinase deficiency.

GLUCOSE-6-PHOSPHATE DEHYDROGENASE AND RED CELL PYRUVATE KINASE DEFICIENCY IN NEONATAL JAUNDICE CASES IN EGYPT

Glucose-6-phosphate dehydrogenase (G6PD) deficiency can lead to acute hemolytic anemia, chronic nonspherocytic hemolytic anemia, and neonatal jaundice. Neonatal red cell pyruvate kinase (PK) deficiency may cause clinical patterns, ranging from extremely severe hemolytic anemia to moderate jaundice. The authors aimed at studying the prevalence of G6PD and PK deficiency among Egyptian neonates with pathological indirect hyperbilirubinemia in Cairo. This case-series study included 69 newborns with unconjugated hyperbilirubinemia. All were subjected to clinical history, laboratory investigations, e.g., complete blood counts, reticulocytic counts, direct and indirect serum bilirubin levels, Coombs tests, qualitative assay of G6PD activity by methemoglobin reduction test, and measurement of erythrocytic PK levels. The study detected 10 neonates with G6PD deficiency, which means that the prevalence of G6PD deficiency among Egyptian neonates with hyperbilirubinemia is 14.4% (21.2% of males). G6PD deficiency was significantly higher in males than females (P = .01). The authors detected 2 cases with PK deficiency, making the prevalence of its deficiency 2.8%. These data demonstrate that G6PD deficiency is an important cause for neonatal jaundice in Egyptians. Neonatal screening for its deficiency is recommended. PK deficiency is not a common cause of neonatal jaundice. However, this needs further investigation on a larger scale.

Coexistence of Congenital Red Cell Pyruvate Kinase Deficiency and Hereditary Stomatocytosis.

We describe a case of chronic hemolytic anemia due to the co-presence of pyruvate kinase (PK) deficiency and Hereditary Stomatocytosis (HSto). The propositus was a 30 years old adopted male with no known family history; he had severe neonatal jaundice requiring exchange transfusion, followed by a life-long history of moderate to severe chronic hemolytic anemia (Hb 7–10 g/dL), with jaundice and splenomegaly. At the age of 6 months hemoglobin screening was made and a beta trait was found. At the age of 20 splenectomy and cholecystectomy were performed. Surgery resulted in an increase of 1.5 g/dL in haemoglobin, and in a conspicuous rise of reticulocytes (from 125×109/L to 562×109/L). Two thrombotic events occurred thereafter, the former 6 days after surgery, and the latter two years later, during a toxoplasmosis infection. At the time of the study Hb was 10.8 g/dL, MCV 82.2 fL, reticulocytes 562×109/L, unconjugated bilirubin 2.19 mg/dL, LDH 335 U/L, haptoglobin <20 mg/dL, serum ferritin 342 ng/mL and transferrin saturation 71%. The peripheral blood smear examination showed the presence of echinocytes (13%), stomatocytes (11%), acantocytes (10%), schistocytes (7%), elliptocytes (6%), spherocytes (4%), target cells (4%) and a few erythroblasts. Erythrocyte osmotic fragility was decreased; screening test for unstable hemoglobins and sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) of red cell membrane gave normal results. The study of the most important red cell enzymes revealed reduced PK activity (6.0 UI/gHb, normal range 11.1–15.59 UI/gHb) and thermal stability (43%, normal range 57–100%). Direct sequencing of PK-LR gene showed a compound heterozygosity for mutation 1456T (Arg486Trp) and the new variant −73g>c. Mutation −73g>c occurs in the most proximal of the four GATA motifs in the R-type promoter region and possibly result in a decrease of mRNA synthesis, as already reported for the variant −72a>g (Manco et al, 2000). Molecular analysis of HFE gene showed heterozygosity for H63D mutation. The history of post splenectomy thrombosis and the presence of stomatocytes in peripheral blood smear prompted us to investigate for the coexistence of hereditary stomatocytosis. The determination of plasma potassium and sodium concentration revealed an increase in intracellular sodium (16.3 mmol/LRBC, reference range 5.0–12.0) and a decrease in intracellular potassium (74.73 mmol/LRBC, reference range 90–103), suggestive for a diagnosis of dehydrated HSto, or hereditary xerocytosis. This defect likely accounts for the thrombophilic state in this case, since HSto is known to be associated with hypercoagulability, particulary after splenectomy.

Transgenic rescue of hemolytic anemia due to red blood cell pyruvate kinase deficiency

Red blood cell pyruvate kinase (R-PK) deficiency is the most common glycolytic enzyme defect associated with hereditary non-spherocytic hemolytic anemia. Cases with the most severe deficiency die in the peri- or neonatal period and no specific therapy exists at present. To test whether the targeted overexpression of the normal R-PK gene in erythroid cells could reduce hemolysis in R-PK mutant mice, we performed a genetic rescue study using human R-PK transgenic mice. Human R-PK promoter driven with human mLCR of the human b-globin locus was used for the erythroid-specific expression of human R-PK in murine erythrocytes. The transgenic lines were mated with homozygous R-PK mutant mice and subsequently backcrossed. Mutant homozygotes with the mLCR-R-PK transgene were examined for any therapeutic effects of transgene expression. Two PK transgenic lines, hRPK_lo and hRPK_hi, were obtained. R-PK activity of the transgenic mice reached as high as three times that of the animals with the endogenous PK gene. Overexpression of human R-PK in the homozygous mutant mice successfully reduced hemolytic anemia. Improvements of hemolysis were evaluated by hemoglobin concentration, reticulocyte count, and spleen weight, which showed significant correlations with the levels of expression of the transgene. Recovery from metabolic disturbance in mutant red blood cells was shown as normalized concentrations of the glycolytic intermediates upstream of PK. In addition, there was a remarkable negative correlation between R-PK activity and the number of TUNEL-positive erythroid progenitors in the spleen. These results indicate that overexpression of the wild-type PK gene in mutant erythroid cells ameliorates both erythroid apoptosis and the shortened red blood cell lifespan observed in PK mutant mice. It is likely that the level of transgene expression required to achieve evident therapeutic effects should be equivalent to or more than that of the endogenous PK gene. This gene-addition strategy may be suitable for clinical application if there is a high level of transgene expression of R-PK in erythroid progenitors/red blood cells.

Pyruvate kinase deficiency: The genotype-phenotype association

Red cell pyruvate kinase (PK) deficiency is the most frequent enzyme abnormality of glycolysis causing chronic non-spherocytic haemolytic anaemia. The disease is transmitted as an autosomal recessive trait, clinical symptoms usually occurring in compound heterozygotes for two mutant alleles and in homozygotes. The severity of haemolysis is highly variable, ranging from very mild or fully compensated forms to life-threatening neonatal anaemia necessitating exchange transfusions. Erythrocyte PK is synthesised under the control of the PK-LR gene located on chromosome 1. One hundred eighty different mutations in PK-LR gene, mostly missense, have been so far reported associated to PK deficiency. First attempts to delineate the genotype-phenotype association were mainly based on the analysis of the enzyme's three-dimensional structure and the observation of the few homozygous patients. More recently, the comparison of the recombinant mutants of human red cell PK with the wild-type enzyme has enabled the effects of amino acid replacements on the enzyme molecular properties to be determined. However, the clinical manifestations of red cell enzyme defects are not merely dependent on the molecular properties of the mutant protein but rather reflect the complex interactions of additional factors, including genetic background, concomitant functional polymorphisms of other enzymes, posttranslational or epigenetic modifications, ineffective erythropoiesis and differences in splenic function.

Red cell pyruvate kinase deficiency: 17 new mutations of the PK‐LR gene

The PK-LR gene was studied in 23 patients with congenital haemolytic anaemia associated with erythrocyte pyruvate kinase (PK) deficiency. Twenty-seven different mutations were detected among the 42 mutated alleles identified: 19 missense mutations, four splice site mutations and one nonsense, one single base deletion and two large deletions. Seventeen of them (107G, 278T, 403T, 409A, 661A, 859C, 958A, 1094T, 1190T, 1209A, 1232C, 1369G, 507A, IVS9 -1c, IVS9 +43c [corrected] del C224, del 5006bp IVS3--> nt 1431) were new. Although all the exons, the flanking regions and the promoter were sequenced in all cases, we failed to detect the second expected mutation in four subjects. To correlate genotype to phenotype, the molecular results were related to the biochemical properties of the mutant enzymes by an analysis of the three-dimensional structure of erythrocyte PK. The new mutant 409A, found in association with the large deletion of 5006 bp in a newborn baby who died soon after birth, was functionally characterized by mutagenesis and in vitro expression of the protein to investigate its contribution in the severity of the clinical pattern. However, the biochemical data obtained for the mutant enzyme cannot explain the severe anaemia found in the PK-deficient patient hemizygous for this mutation.

Red cell pyruvate kinase deficiency: molecular and clinical aspects

Red cell pyruvate kinase (PK) deficiency is the most frequent enzyme abnormality of the glycolytic pathway causing hereditary non-spherocytic haemolytic anaemia. The degree of haemolysis varies widely, ranging from very mild or fully compensated forms, to life-threatening neonatal anaemia and jaundice necessitating exchange transfusions. Erythrocyte PK is synthesized under the control of the PK-LR gene located on chromosome 1. To date, more than 150 different mutations in the PK-LR gene have been associated with PK deficiency. First attempts to delineate the biochemical and clinical consequences of the molecular defect were mainly based on the observation of the few homozygous patients and on the analysis of the three-dimensional structure of the enzyme. More recently, the comparison of the recombinant mutants of human red cell PK with the wild-type enzyme has enabled the effects of amino acid replacements on the enzyme molecular properties to be determined and help to correlate genotype to clinical phenotype.

Red Cell Pyruvate Kinase Deficiency: Molecular Characterization of 10 New Variants.

PK deficiency is the most common glycolytic enzyme defect associated with chronic non-spherocytic hemolytic anemia. To date about 150 different mutations have been identified in the PK-LR gene. Among them only one large deletion has been described in Gipsy resulting in the loss of exon 11. We report 10 new variants of LR-PK gene in 8 families with pyruvate kinase deficiency. The entire coding region and intronic flanking regions were analyzed by direct sequencing. The results of the molecular analysis are reported in the table:PtOriginHb (g/dL)Tx (n.)PK Activity (IU/gHb)MutationAminoacidic substitutionSDItaly15.1010.6107G / ?Ala36Gly/?TTAustralia8.90nd409A / del5006bp( IVS3-nt 1431)Ala137Thr / del ex 4-11NRItaly9>505.5661A /1209AAsp221Asn /Met403IleNAItaly9>505.3661A /1209AAsp221Asn /Met403IleSAItaly1205.51456T/ 1209AArg486Trp/ Met403IleSCItalyndnd5.61529A/ 859CArg510Gln/ Phe287LeuCMItaly9.5013.61456T/ 958AArg486Trp/ Val320MetPSItaly13.2081094T /?Lys365Met /?VRItaly10.5010.71706A / ?Arg569Gln /?GRGuinea14.407.61269A/ IVS9+43cSplice site/?Ref. Values12.2–1611.1–15.5Mutations reported in bold are new. By comparing the amino acids sequences among several species (cat M1, chicken M, rat L, yeast and human), we found that mutations 661A, 859C, 958A, 1094T and 1209A involve highly conserved residues. Mutation 1209A when present in association with 661A (cases NA and NR) results in a severe clinical pattern with need of transfusion support, whereas in compound heterozygosity with 1456T (case SA, mother of NA and NR) is associated with a less severe clinical pattern. The variant 1706A was found in a patient carrying the polymorphism 1705C at the homozygous level; the mutation in association with the polymorphism determines the aminoacidic substitution Arg596Gln.A deletion of 5006 nucleotides extending from intron 3 to the last 3 nucleotides of exon 10 has been found in an Australian baby dead at birth (case TT); the mutation results in a large cDNA deletion encompassing exon 4 and exon11 included. This is the largest abnormality so far detected in LR-PK gene.

Successful bone marrow transplantation in a child with red blood cell pyruvate kinase deficiency

We report the first successful use of BMT for the treatment of RBC pyruvate kinase (PK) deficiency in a boy who developed neonatal jaundice and severe transfusion-dependent hemolytic anemia a few months after birth. He received a BMT at the age of 5 from an HLA-identical sister who has normal PK activity after conditioning with busulfan and cyclophosphamide. The post-transplant course was uneventful. At present, 3 years after transplant, he is 8 years old and has a normal hemoglobin level and normal RBC PK activity without evidence of hemolysis. DNA analysis has confirmed full engraftment.