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.

Clinical outcome and genotype analysis of four Chinese children with pyruvate kinase deficiency.

BACKGROUND: Pyruvate kinase deficiency (PKD) is a rare congenital hemolytic anemia. Here, we summarized the clinical features and laboratory examinations of four Chinese children with PKD and analyze genomic mutations. METHOD: Collected and analyzed the clinical data of all children and their parents and completed the relevant laboratory examinations of all children. Analyzed the sequences of related genes in children by second-generation sequencing technology and verified the suspected mutations in children's family by Sanger sequencing method or second-generation sequencing technology. RESULTS: A total of six mutations in gene PKLR were detected in four cases. Except for c.1510C>T (P1) and c.941T>C (P2 and P4), which had been reported in previous studies, the other four novel gene mutations were reported for the first time, including a rare homozygous mutation with large fragment deletion. All those gene mutations cause changes in the amino acids encoded by the gene, as well as subsequent changes in protein structure or loss of function. CONCLUSION: Compound heterozygous or homozygous mutations in the coding region of PKLR gene are the causes of PKD in these four Chinese children. The second-generation sequencing technology is an effective means to diagnose PKD. The mutations of c.457-c.462delATCGCC, c.1297T>C, c.1096C>T and Exon4-10del of PKLR reported in this article have not been included in the Thousand Genome Database, dbSNP(v138) and ExAC Database. The PKLR gene mutations found in these children with PKD can provide references for further research of the genetic characteristics of PKD and subsequent gene therapy.

Association of PKLR gene copy number, expression levels and enzyme activity with 2,3,7,8-TCDD exposure in individuals exposed to Agent Orange/Dioxin in Vietnam.

2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD) is the most toxic congener of dioxin and has serious long-term effects on the environment and human health. Pyruvate Kinase L/R (PKLR) gene expression levels and gene variants are associated with pyruvate kinase enzyme deficiency, which has been identified as the cause of several diseases linked to dioxin exposure. In this study, we estimated PKLR gene copy number and gene expression levels using real-time quantitative PCR (RT-qPCR) assays, genotyped PKLR SNP rs3020781 by Sanger sequencing, and quantified plasma pyruvate kinase enzyme activity in 100 individuals exposed to Agent Orange/Dioxin near Bien Hoa and Da Nang airfields in Vietnam and 100 healthy controls. The means of PKLR copy numbers and PKLR gene expression levels were significantly higher, while pyruvate kinase enzyme activity was significantly decreased in Agent Orange/Dioxin-exposed individuals compared to healthy controls (P < 0.0001). Positive correlations of PKLR gene copy number and gene expression with 2,3,7,8-TCDD concentrations were observed (r = 0.2, P = 0.045 and r = 0.54, P < 0.0001, respectively). In contrast, pyruvate kinase enzyme activity was inversely correlated with 2,3,7,8-TCDD concentrations (r = -0.52, P < 0.0001). PKLR gene copy number and gene expression levels were also inversely correlated with pyruvate kinase enzyme activity. Additionally, PKLR SNP rs3020781 was found to be associated with 2,3,7,8-TCDD concentrations and PKLR gene expression. In conclusion, PKLR copy number, gene expression levels, and pyruvate kinase enzyme activity are associated with 2,3,7,8-TCDD exposure in individuals living in Agent Orange/Dioxin-contaminated areas.

Pyruvate kinase deficiency mimicking congenital dyserythropoietic anemia type I.

BACKGROUND: Pyruvate kinase (PK) deficiency is the most common enzyme abnormality in the glycolytic pathway. Here, we describe two siblings with PK deficiency that mimicked congenital dyserythropoietic anemia (CDA) type I. CASE: The siblings were referred to our hospital for evaluation of anemia when they were newborns. Their PK enzyme activities were normal. Their bone marrow aspirations and electron microscopies showed CDA-like findings. A CDA panel with next-generation sequencing showed no mutation. Though their PK enzyme levels were normal, a molecular study of the PKLR gene showed a homozygous variant c.1623G > C (p.Lys541Asn) in exon 12 of our patients. CONCLUSIONS: Although the diagnosis of pyruvate kinase deficiency is difficult, it can be confused with many other diagnoses. Bone marrow findings of these cases are similar to congenital dyserythropoietic anemia. In patients with normal pyruvate kinase enzyme levels, the diagnosis cannot be excluded and genetic analysis is required.

Genetic Diagnosis of Pyruvate Kinase Deficiency in Undiagnosed Iranian Patients with Severe Hemolytic Anemia, using Whole Exome Sequencing.

BACKGROUND: After ruling out the most common causes of severe hemolytic anemia by routine diagnostic tests, certain patients remain without a diagnosis. The aim of this study was to elucidate the genetic cause of the disease in these patients using next generation sequencing (NGS). METHODS: Four unrelated Iranian families including six blood transfusion dependent cases and their parents were referred to us from a specialist center in Tehran. There was no previous history of anemia in the families and the parents had no abnormal hematological presentations. All probands presented severe congenital hemolytic anemia, neonatal jaundice and splenomegaly. Common causes of hemolytic anemia were ruled out prior to this investigation in these patients and they had no diagnosis. Whole exome sequencing (WES) was performed in the probands and the results were confirmed by Sanger sequencing and subsequent family studies. RESULTS: We identified five variants in the PKLR gene, including a novel unpublished frameshift in these families. These variants were predicted as pathogenic according to the ACMG guidelines by Intervar and/or Varsome prediction tools. Subsequent family studies by Sanger sequencing supported the diagnosis of pyruvate kinase deficiency (PKD) in six affected individuals and the carrier status of disease in their parents. CONCLUSION: These findings show that PKD is among the rare blood disorders that could remain undiagnosed or even ruled out in Iranian population without performing NGS. This could be due to pitfalls in clinical, hematological or biochemical approaches in diagnosing PKD. Furthermore, genotyping PKD patients in Iran could reveal novel mutations in the PKLR gene.

A Sri Lankan girl with a new genetic variant in the PKLR gene causing pyruvate kinase deficiency: a case report.

BACKGROUND: Erythrocyte pyruvate kinase is expressed under the control of the PKLR gene located on chromosome 1q21. Pyruvate kinase catalyzes the final steps of the glycolytic pathway and creates 50% of the red cell total adenosine triphosphate. Pyruvate kinase deficiency is the commonest glycolytic defect causing congenital non-spherocytic hemolytic anemia inherited in an autosomal recessive trait in which homozygotes and compound heterozygotes are common. Over 200 mutations have been described in patients with pyruvate kinase deficiency. This case report identifies a new pathogenic variant in PKLR gene detected in a patient with severe pyruvate kinase deficiency. CASE PRESENTATION: A Sri Lankan Sinhalese girl who developed neonatal anemia and jaundice within 24 hours of birth with mild hepatomegaly. She was from a nonconsanguineous marriage and had two siblings who had no hematological disorders. She had repeated admissions due to similar illnesses and at the age of 8 years was found to have pyruvate kinase deficiency associated with a novel homozygous pathogenic variant c.507+1delG in the PKLR gene. CONCLUSIONS: A novel genetic variant in PKLR gene, consistent with pyruvate kinase deficiency, was detected in a Sri Lankan girl. This genetic variant may be specific to the Asian population and requires further studies.

Novel mutations associated with pyruvate kinase deficiency in Brazil.

BACKGROUND: Pyruvate kinase deficiency is a hereditary disease that affects the glycolytic pathway of the red blood cell, causing nonspherocytic hemolytic anemia. The disease is transmitted as an autosomal recessive trait and shows a marked variability in clinical expression. This study reports on the molecular characterization of ten Brazilian pyruvate kinase-deficient patients and the genotype-phenotype correlations. METHOD: Sanger sequencing and in silico analysis were carried out to identify and characterize the genetic mutations. A non-affected group of Brazilian individuals were also screened for the most commonly reported variants (c.1456C>T and c.1529G>A). RESULTS: Ten different variants were identified in the PKLR gene, of which three are reported here for the first time: p.Leu61Gln, p.Ala137Val and p.Ala428Thr. All the three missense variants involve conserved amino acids, providing a rationale for the observed enzyme deficiency. The allelic frequency of c.1456C>T was 0.1% and the 1529G>A variant was not found. CONCLUSION: This is the first comprehensive report on molecular characterization of pyruvate kinase deficiency from South America. The results allowed us to correlate the severity of the clinical phenotype with the identified variants.

Novel mutations associated with pyruvate kinase deficiency in Brazil

Abstract Background: Pyruvate kinase deficiency is a hereditary disease that affects the glycolytic pathway of the red blood cell, causing nonspherocytic hemolytic anemia. The disease is transmitted as an autosomal recessive trait and shows a marked variability in clinical expression. This study reports on the molecular characterization of ten Brazilian pyruvate kinase-deficient patients and the genotype-phenotype correlations. Method: Sanger sequencing and in silico analysis were carried out to identify and characterize the genetic mutations. A non-affected group of Brazilian individuals were also screened for the most commonly reported variants (c.1456C>T and c.1529G>A). Results: Ten different variants were identified in the PKLR gene, of which three are reported here for the first time: p.Leu61Gln, p.Ala137Val and p.Ala428Thr. All the three missense variants involve conserved amino acids, providing a rationale for the observed enzyme deficiency. The allelic frequency of c.1456C>T was 0.1% and the 1529G>A variant was not found. Conclusion: This is the first comprehensive report on molecular characterization of pyruvate kinase deficiency from South America. The results allowed us to correlate the severity of the clinical phenotype with the identified variants.