Anemia, Hemolytic, Congenital Nonspherocytic ; Erythrocytes ; Humans ; Pyruvate Kinase/deficiency* ; Pyruvate Metabolism, Inborn Errors

The RBC enzyme deficiencies causing hereditary hemolytic anemia (HHA) can be divided into three groups: those participating in the glycolytic (E-M) pathway; those involved with the maintenance of a high ratio of reduced to oxidized glutathione; one enzyme in the nucleotide degradation and salvage pathway. Although RBC enzyme deficiencies causing HHA are rare, 3 of the 15 kinds of important and relatively frequently reported enzyme deficiencies such as pyruvate kinase, glucose-6-phosphate-dehydrogenase and pyrimidine-5'-nucleotidase deficiencies are briefly reviewed. The molecular genetics, clinical symptoms, diagnosis and therapeutic approaches of each enzyme deficiencies are summerized. As these enzyme deficiencies are reported throughout the world as well as in Korea with the identification of the mutations, considering a broad spectrum of etiologies for the diagnosis of HHA seems to be warranted.
Anemia, Hemolytic, Congenital ; Erythrocytes ; Glucosephosphate Dehydrogenase Deficiency ; Korea ; Molecular Biology ; Pyruvate Kinase

OBJECTIVETo screen potential mutation and explore the underlying mechanism for a consanguineous pedigree featuring pyruvate kinase (PK) deficiency.

METHODSThe red blood cell pyruvate kinase activities of all family members were detected. All the exons and intron-exon boundaries of the PKLR gene for the proband were amplified and analyzed by direct sequencing. Restriction endonuclease enzymes were used to identify the presence of mutations of all family members.

RESULTSThe pyruvate kinase activities were 5.89 U/g Hb in the proband, 3.45, 6.54, 8.87, 7.89, 9.32 U/g Hb in his younger sister, father, mother, grandmother and elder aunt, respectively. The homozygous missense mutation of T>C transition at position 941 in exon 7 of PKLR gene resulted to a Ile314Thr substitution in the proband, and mutant alleles were identified at the level of RNA transcript by cDNA sequence analysis. His younger sister was also homozygous for Ile314Thr. Heterozygosity for Ile314Thr was confirmed in his grandmother, parents and elder aunt.

CONCLUSIONIle314Thr homozygous missense mutation in exon 7 of PKLR is the molecular mechanism of pyruvate kinase deficiency in this family.

Anemia, Hemolytic, Congenital Nonspherocytic ; genetics ; Child, Preschool ; Female ; Humans ; Male ; Pedigree ; Point Mutation ; Pyruvate Kinase ; deficiency ; genetics ; Pyruvate Metabolism, Inborn Errors ; genetics

OBJECTIVETo explore the molecular mechanism of erythrocyte pyruvate kinase deficiency (PKD).

METHODSTargeted sequence capture and next-generation sequencing (NGS) were used to detect the regions of exon and exon-intron boundarie of PKLR gene in a clinical suspected PKD patient. The protein function of mutant gene was forecasted by the SIFT and PolyPhen-2 databank, after the mutation of PKLR gene in the patient was detected by the NGS technology, its genotype was confirmed by Sanger sequencing.

RESULTSThe patient was found to have peculiar double heterozygous mutations: 661 G>A (Asp221Asn) of exon 5 and 1528 C>T (Arg510Ter) of exon 10, resulting in amino acid substitution Asp221Asn and Arg510Ter, these mutations were also further confirmed by Sanger sequencing. The complex mutations were infrequent and each of them was able to cause diseases.

CONCLUSIONThe complex mutations of both 661 G>A and 1528 C>T of PKLR gene are the molecular mechanism of PKD. Simultaneous existance of above-mentioned complex mutations in PDK patient was never been previously reported at home and abroad.

Anemia, Hemolytic, Congenital Nonspherocytic ; genetics ; Exons ; Genotype ; High-Throughput Nucleotide Sequencing ; Humans ; Introns ; Mutation ; Pyruvate Kinase ; deficiency ; genetics ; Pyruvate Metabolism, Inborn Errors ; genetics

OBJECTIVETo evaluate the feasibility of genetic and prenatal diagnosis for a family affected with pyruvate kinase deficiency (PKD).

METHODSTargeted sequence capture and high-throughput sequencing technology was used to detect the exons and exon-intron boundaries of the PKLR gene in a clinically suspected PKD patient. Meanwhile, the genotype of the pedigree was validated by Sanger sequencing. Prenatal genetic diagnosis was performed by amniotic fluid sampling after genotype of the mother of the proband was determined.

RESULTSThe proband was found to harbor double heterozygous mutations, c.661G>A (Asp221Asn) and c.1528C>T (Arg510Ter), which resulted in amino acid substitution Asp221Asn and Arg510Ter. Such mutations were confirmed by Sanger sequencing. The mother and father of the proband were detected to have respectively carried c.1528C>T (Arg510Ter) and c.661G>A (Asp221Asn) mutation. The fetus was found to have carried the same mutations as the proband. Following selected abortion, analysis of fetal tissue was consistent with the result of prenatal diagnosis.

CONCLUSIONThe compound mutations of c.661G>A and c.1528C>T of PKLR gene probably underlie the PKD in the family. Prenatal diagnosis of the mutations analysis can facilitate detection of affected fetus in time.

Adult ; Anemia, Hemolytic, Congenital Nonspherocytic ; embryology ; enzymology ; genetics ; Base Sequence ; Child, Preschool ; DNA Mutational Analysis ; Exons ; Female ; Genotype ; Humans ; Male ; Molecular Sequence Data ; Mutation ; Pedigree ; Pregnancy ; Prenatal Diagnosis ; Pyruvate Kinase ; deficiency ; genetics ; metabolism ; Pyruvate Metabolism, Inborn Errors ; embryology ; enzymology ; genetics