Pyruvate kinase deficiency and severe congenital hemolytic anemia in a double heterozygous patient with paternal transmission of an early germ‐line <i>de novo</i> mutation
Authors: María Del Mar Mañú‐Pereira, Eva Gonzalez‐Roca, Wouter W. van Solinge, Esther Llaudet‐Planas, Julián Sevilla, Laura Montllor, Anna Mensa‐Vilaro, Hans Kristian Ploos van Amstel, Richard van Wijk, JoanLluis Vives‐Corrons
Journal: American Journal of Hematology (2015)

To the Editor: Red blood cell (RBC) pyruvate kinase (PK) deficiency (MIM 609712) is the most common glycolytic defect leading to chronic nonspherocytic hemolytic anemia (CNSHA). PK deficiency is a rare autosomal recessive disease with a worldwide distribution and an estimated prevalence of about 1:20,000 in the general white population 1. Patients with PK deficiency are homozygous for two identical mutated alleles or, more frequently, biallelic carriers of two different mutations at the PKLR locus, with a highly variable phenotype, ranging from severe CNSHA since birth, to moderate anemia with exacerbation during infection or pregnancy, or compensated hemolysis with slight or no anemia 2. Genetic studies of deficient PKLR gene have shown prevalent mutations according to patient's origin 3 and more than 240 mutations have been described so far (www.lovd.nl/pklr). De novo PKLR mutations are extremely rare and occur in about 3% of patients. In none of these cases the transmission of an early germ-line PKLR mutation been ruled out. Herein, we report a case of PK deficiency because of biallelic mutation, where clinical diagnosis was difficult because of the effect of RBCs transfusions and the complex inheritance pattern. The propositus was an 18 months old boy that had presented with severe neonatal CNSHA and jaundice requiring phototherapy and subsequent blood transfusions every 3–4 weeks. For the study, the patient's peripheral blood was obtained just before a blood transfusion, and genetic studies were performed in both patient's progenitors and father grandparents. This study was approved by the medical ethics committee of the Hospital Clinic of Barcelona and in accordance with the Helsinki declaration, and written informed consents were also obtained from all patients' relatives. RBC enzyme activities of glycolysis and oxidative metabolism were measured according to standard procedures 4, and to relate PK activity to the mean age of the RBCs, the PK/HK ratio was calculated. PK and also HK activities are strongly RBC age dependent, and in our patient the highly increased young RBC populations was associated with normal PK activity and decreased PK/HK ratio, as usually observed in patients with PK deficiency and severe regenerative anemia (Table 1). Electropherograms of nucleotide c.1168 of PKLR gene (black arrows) obtained by Sanger sequencing. A: Analysis of genomic DNA obtained from peripheral blood of patient, his parents, and his grandparents. B: Analysis of genomic DNA obtained from different samples of patient's father: mucosal swab, urine, and sperm. Genomic DNA from the patient and his relatives was isolated from whole blood leukocytes using a QIAamp DNA Blood Mini kit (Qiagen, Germany). PKLR promoter, the 12 exons, and flanking intronic sequences were amplified by polymerase chain reaction (PCR) and purified using a QIAquick PCR Purification kit (Qiagen). Sequencing run was performed on a 454 GS Junior (Roche, Switzerland) using the GS Junior Titanium Sequencing kits (Roche, Switzerland). Results were analyzed using the software Amplicon Variant Analyzer software (Roche, Switzerland). DNA analysis of patient's PKLR revealed that he was a compound heterozygous for two known mutations of PKLR gene: c.359C>T (p.Ser120Phe) mutation in exon 4; and c.1168G>A (p.Asp390Asn) mutation in exon 9 (www.lovd.nl/pklrwww.pklrmutationdatabase.com). Family studies demonstrated the presence of the c.359C>T mutation in patient's mother and a normal genotype in the father (Fig. 1A). Allelic inheritance analysis was performed to confirm familial relationship and exclude the possibility of sample mix-up. It included the analysis of 10 highly polymorphic genomic markers on chromosomes X, Y, 3, 4, 5, 7, 8, 12, 13, 18 and 21. Since an in vitro fecundation process, carried out in the family after diagnosis, showed a c.1168 G>A change in five out of the nine embryos obtained, it was hypothesized that this mutation had arisen de novo in the father during his embryonic life. To investigate this possibility a massive parallel sequence analysis (NGS) of the region encompassing nucleotide c.1168 of PKLR was carried out on paternal genomic DNA from blood cells, mucosal swabs, 24 hours urine, and sperm samples. Exon 9 of PKLR, harboring the c.1168G>A mutation, was amplified using a two-step PCR procedure as described by Izawa et al 5. Library preparation, emulsion PCR, and sequencing were performed according to the manufacturer's instructions. Results of this study demonstrated that the c.1168G>A mutation was present in all the analyzed tissues, but at different levels of abundance: the highest percentage was found in sperm cells (19.3%) and the lowest, in blood cells (6.9%). Since the sequencing of the same region performed on peripheral blood genomic DNA from paternal grandparents, was normal, the de novo origin of the c.1168G>A in the father was confirmed. Conventional Sanger sequencing did not clearly identify the mutant allele in the father's blood cells DNA (Fig. 1A) because it was less than 10%. However, it was detected in the DNA from sperm, mucosal swab, and urine samples (Fig. 1B) because of their higher abundance of the mutant allele. This is not surprising in view of the limits of sensitivity of Sanger sequencing. Moreover, the low abundance of blood cells with the mutant PKLR allele may also explain the normal PK activity found in the father RBCs. These results allow us to assume that the c.1168G>A mutation has appeared during progenitor's early embryonic life, just before the separation of germ line cells from the somatic cells. Therefore, the patient's father, being heterozygous for the mutation in some of his sperm cells, has to be considered the heterozygous carrier that has transmitted the PK c.1168G>A mutation to his son. The probability of the father transmitting the mutant allele is 19%, whereas 50% of the mature oocytes of the mother will harbor her mutant allele. Therefore, the expected recurrence rate for a second compound heterozygous child is, in this case, approximately 10%. We don't know whether the different abundance of mutant cells in different tissues resulted from the number of founder cells that contributed to form those tissues, or to subsequent selective forces to which cells with different PK levels (about 50% versus 100%) might have been sensitive. Up to now, vertical transmission of de novo germ-line mutations have been described for several dominant inherited diseases, but for only one recessive disease 6. We conclude that in patients with suspected "de novo" PK, the use of NGS gene technology is a very useful tool for demonstrating mosaicism and, in turn, for a correct genetic counselling. This study has been partially funded by the PI14/00772 project, part of the Spanish R+D+I National Plan and funded by the ISCIII-Subdirección General de Evaluación and the Fondo Europeo de Desarrollo Regional (FEDER) and supported by the logistic contribution of the European Network for Rare and Congenital Anaemias (ENERCA) that has facilitated the connection and information exchange between the centers participating in this study (www.enerca.org). The authors are indebted to Prof. Lucio Luzzatto from the Istituto Toscano Tumori (University of Genova) for his critical review and kindly help in preparing this manuscript. María Del Mar Mañú-Pereira,1 Eva Gonzalez-Roca,2 Wouter W. van Solinge,3 Esther Llaudet-Planas,1 Julián Sevilla,4 Laura Montllor,1 Anna Mensa-Vilaro,2 Hans Kristian Ploos van Amstel,5 Richard van Wijk,3 and Joan-Lluis Vives-Corrons1* 1Red Cell Pathology Unit, Hospital Clínic, IDIBAPS, University of Barcelona, Spain; 2Immunology Department, Hospital Clínic, IDIBAPS, Barcelona, Spain; 3Department of Clinical Chemistry and Haematology, University Medical Center Utrecht, Utrecht, the Netherlands; 4Department of Pediatrics, Hospital Niño Jesús, Madrid, Spain; 5Department of Medical Genetics, University Medical Center Utrecht, Utrecht, the Netherlands