Analysis and prenatal diagnosis of PKLR gene mutations in a family with pyruvate kinase deficiency / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To evaluate the feasibility of genetic and prenatal diagnosis for a family affected with pyruvate kinase deficiency (PKD).</p><p><b>METHODS</b>Targeted 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.</p><p><b>RESULTS</b>The 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.</p><p><b>CONCLUSION</b>The 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.</p>

A PKLR Gene Novel Complex Mutation in Erythrocyte Pyruvate Kinase Deficiency Detected by Targeted Sequence Capture and Next Generation Sequencing / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To explore the molecular mechanism of erythrocyte pyruvate kinase deficiency (PKD).</p><p><b>METHODS</b>Targeted 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.</p><p><b>RESULTS</b>The 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.</p><p><b>CONCLUSION</b>The 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.</p>

Analysis of a pyruvate kinase deficiency consanguineous pedigree caused by Ile314Thr homozygous mutation / 中华血液学杂志

<p><b>OBJECTIVE</b>To screen potential mutation and explore the underlying mechanism for a consanguineous pedigree featuring pyruvate kinase (PK) deficiency.</p><p><b>METHODS</b>The 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.</p><p><b>RESULTS</b>The 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.</p><p><b>CONCLUSION</b>Ile314Thr homozygous missense mutation in exon 7 of PKLR is the molecular mechanism of pyruvate kinase deficiency in this family.</p>

Erythrocyte glucose-6-phosphate dehydrogenase and pyruvate kinase activities in hemoglobin H disease.

Erythrocyte glucose-6-phosphate dehydrogenase (G6PD) and pyruvate kinase (PK) activities were studied in hemoglobin H (HbH) patients by spectrophotometric method, cytochemical method and the methemoglobin reduction (MR) test for the detection of heterozygous G6PD deficiency. G6PD deficiency was found in 7 of 64 cases (10.9%), including 3 cases of genotype alpha 1/alpha 2 and 4 cases of genotype alpha 1/CS. None of the HbH patients was found to be PK-deficient. Spectrophotometrically determined G6PD and PK activities were significantly higher in HbH patients than in normals (p less than 0.001), whereas the MR test yielded a significantly lower percentage of residual methemoglobin in HbH patients than in normals (p less than 0.05). All three methods were efficient in the detection of hemizygous G6PD deficiency in HbH patients, but not in G6PD-deficient females.