First-trimester prenatal diagnosis of pyruvate kinase deficiency in an Indian family with the pyruvate kinase-Amish mutation Genetics and Molecular Research 6 (2): 470-475 (2007) FUNPEC-RP www.funpecrp.com.br First-trimester prenatal diagnosis of pyruvate kinase deficiency in an Indian family with the pyruvate kinase-Amish mutation P.S. Kedar1, S. Nampoothiri2, S. Sreedhar2 , K. Ghosh1, K. Shimizu3, H. Kanno4 and R.B. Colah1 1Institute of Immunohaematology, Indian Council of Medical Research, K.E.M. Hospital Campus, Parel, Mumbai, India 2Department of Pediatric Genetics, Amrita Institute of Medical Sciences and Research Center, Ernakulam, Cochin, India 3Department of Bioscience and Bioinformatics, Kyushu Institute of Technology, Iizuka, Fukuoka, Japan 4Department of Transfusion Medicine and Cell Processing, Tokyo Women’s Medical University, Tokyo, Japan Corresponding author: R.B. Colah E-mail: colahrb@gmail.com AbStrAct. Pyruvate kinase (PK) deficiency is a rare red cell gly- colytic enzymopathy. The purpose of the present investigation was to offer prenatal diagnosis for PK deficiency to a couple who had a pre- vious child with severe enzyme deficiency and congenital non-sphe- rocytic hemolytic anemia. PK deficiency was identified in the family by assaying the enzyme activity in red cells. Chorionic villus sampling was performed in an 11-week gestation and the mutation was located in exon 10 of the PKLR gene characterized by polymerase chain re- Genet. Mol. Res. 6 (2): 470-475 (2007) Received December 18, 2006 Accepted June 05, 2007 Published June 30, 2007 Case Report Genetics and Molecular Research 6 (2): 470-475 (2007) www.funpecrp.com.br P.S. Kedar et al. 471 INTRODUCTION Red cell pyruvate kinase (PK, EC.2.7.1.40) deficiency is the most common en- zyme abnormality in the Embden Meyerhoff pathway of glycolysis in humans. PK defi- ciency is associated with hereditary non-spherocytic hemolytic anemia and is transmitted as an autosomal recessive disorder (Jacobasch and Rapoport, 1996). The clinical severity of this disorder varies widely, ranging from a mildly compensated anemia to severe child- hood anemia, though in some cases it does not manifest itself until adulthood. Affected newborns usually present unconjugated hyperbilirubinemia and may require exchange transfusion. PK deficiency was first described by Valentine et al. in 1961, and since then, approximately 550 cases have been described in the literature. The human PKLR gene spans 2060 bp of DNA located on the short arm of chromosome 1 and comprises 12 exons (Pissard et al., 2006). Prenatal testing for pyruvate kinase deficiency is sometimes requested by parents, who already have an affected child. Prenatal diagnosis can be done accurately by analyz- ing fetal DNA for the mutation causing the enzyme deficiency. We had earlier screened for mutations in the PKLR gene in a nine-year-old child with severe PK deficiency and identi- fied a homozygous GA substitution at nucleotide 1436, changing arginine to histidine at amino acid residue 479. Both parents were heterozygous for this mutation. This muta- tion has been reported previously among the Old Order Amish of Pennsylvania (Bowman et al., 1965) and the molecular defect was identified (Kanno et al., 1994), where a GA transition at nucleotide 1436 was found. Soon after the initial diagnosis, the mother of the propositus became pregnant. After genetic counseling, the family requested prenatal diagnosis, which was possible as the mutations causing PK deficiency in this family were already identified. CASe RepORT The propositus presented neonatal jaundice at birth and had received an exchange transfusion. He had been transfused 30 times until the age of 8 years, but no diagnosis was action and using restriction endonuclease digestion with the MspI en- zyme, which was confirmed by DNA sequencing on the ABI 310 DNA sequencer. Both the parents were heterozygous for the 1436GA [479 ArgHis] mutation in exon 10 and the proband was homozygous for this mutation. The fetus was also heterozygous for this mutation and the pregnancy was continued. Prenatal diagnosis allowed the parents with a severely affected child with PK deficiency to have the reproductive choice of having the fetus tested in a subsequent pregnancy. Key words: Pyruvate kinase deficiency, Pyruvate kinase-Amish, Neonatal jaundice, Prenatal diagnosis, India Genetics and Molecular Research 6 (2): 470-475 (2007) www.funpecrp.com.br Pyruvate kinase deficiency in an Indian family 472 established. At 9 years, he underwent splenectomy and subsequently was maintaining a hemoglobin level between 8 and 9 g/dL. At that time, he was referred to us for diagnosis as a case of unexplained non-spherocytic hemolytic anemia. At the time of investigation, his Hb level was 9.5 g/dL and reticulocyte count was 28%. Red blood cell PK activity was assayed according to the standard procedure (Beutler, 1984), and PK was found to be markedly reduced (1.8 IU/g Hb at 30°C) with a 3- to 4-fold elevation in 2,3-diphospho- glycerate (2,3-DPG). 2,3-DPG level was determined by the chromotropic acid method (Ea- ton et al., 1969). PK deficiency causes accumulation of few glycolytic intermediates such as phosphoenol pyruvate, 2-phosphoglycerate, 3-phosphoglycerate, and 2,3-DPG in the red cell. Several previous investigators have also reported a good correlation between in- creased levels of 2,3-DPG and PK deficiency (Lestas et al., 1987). The parental red blood cell PK activities (father with 5.85 IU, mother with 5.94 IU/g Hb, at 30°C) were consistent with heterozygosity for PK deficiency. The normal reference range of PK activity was es- tablished using 200 healthy normal control samples and ranged from 11.5 to 16.5 IU/g Hb. DNA analysis showed the propositus to be homozygous and his parents heterozygous for the 1436GA mutation. In the next pregnancy, chorionic villus sampling (CVS) under ul- trasound guidance was performed at 11 weeks gestation, after obtaining informed consent from the couple, and the villus tissue was cleaned under a dissecting microscope and used for molecular analysis. MUTATION ANAlySIS Genomic DNA was extracted from peripheral blood of the parents and the CVS using the Qiagen mini-columns (Hilden, Germany). The 1436GA mutation was identified using re- striction endonuclease digestion with the enzyme MspI (New England Biolabs, UK). A 225-bp DNA fragment of exon 10 of the PKLR gene was amplified by the polymerase chain reaction (PCR) using 10 ng DNA and 200 ng each of primers PK Ex-10F and PK Ex-10R (5’ GAC TAA CAT TCT GGC ACC TG 3’ and 5’ AAG CTC CAT CTG GAC ATT CC 3’, respectively). DNA was initially denatured at 94oC for 4 min followed by 35 cycles of denaturation at 94oC for 20 s, annealing at 58°C for 20 s, and primer extension at 72°C for 20 s, with final extension at 72°C for 5 min. A volume of 20 μL of the PCR product was digested with MspI (2 U) at 37°C overnight. DNA fragments were visualized by UV-transillumination after separation by elec- trophoresis on an 8% polyacrylamide gel and staining with ethidium bromide. DNA sequence analysis of exon 10 was performed on the ABI prism 310 sequencer to confirm the mutation (Kanno et al., 1994). Figure 1A shows the results of restriction enzyme digestion with MspI and the DNA sequencing of the CVS and the proband. The CVS showed fragment sizes of 172 and 132 bp and was heterozygous for the 1436GA mutation. Both parents were also heterozygous for this mutation, while the affected child showed the absence of the 132-bp band. The 225-bp band (Lane 1) corresponds to the undigested PCR product. This is due to heteroduplex forma- tion between normal and mutant DNA resulting in the abolition of the MspI recognition site preventing cleavage. The DNA sequence electropherogram confirmed that the fetus is hetero- zygous (Figure 1C). Figure 1B and D show DNA sequencing of exon 10 in a normal individual and the homozygous child, respectively. Genetics and Molecular Research 6 (2): 470-475 (2007) www.funpecrp.com.br P.S. Kedar et al. 473 DISCUSSION PK deficiency, although rare, is probably the most common cause of hereditary non- spherocytic hemolytic anemia, often causing severe anemia from infancy or early childhood. Prenatal diagnosis by assaying PK activity in fetal blood is not always accurate and molecular analysis is required. The mutations in the PKLR gene resulting in PK deficiency are many and varied; however, once the mutation in the parents and an affected child born earlier is charac- terized, prenatal diagnosis can be easily done in the first trimester of pregnancy by CVS and DNA analysis. We describe the cellular and molecular studies of erythrocyte PK deficiency in a family originating from south India where the mutation in both the parents was the same as in the Dutch Amish population of Pennsylvania. Nucleotide sequencing of the patient’s PK gene showed a point mutation, CGC to CAC, corresponding to nucleotide 1436 from the translational initiation site of the R type PK (R-PK) mRNA, causing a single amino acid substitution from Arg to His at the 479th amino acid residue of the R-PK gene. It has been shown earlier that the substituted Arg Figure 1. Prenatal diagnosis of pyruvate kinase deficiency due to a codon 479 (1436GA) mutation. A. MspI digest of exon 10 of the PKLR gene on 8% polyacrylamide gel electrophoresis. Lane 1, PCR amplified undigested DNA; lane 2, father (heterozygous); lane 3, mother (heterozygous); lane 4, chorionic villus sample (heterozygous); lane 5, proband (homozygous); lane 6, marker VIII (Roche); b. DNA sequencing of exon 10 in a normal individual. c. DNA sequencing of exon 10 of the chorionic villus sample. D. DNA sequencing of exon 10 of the proband. Genetics and Molecular Research 6 (2): 470-475 (2007) www.funpecrp.com.br Pyruvate kinase deficiency in an Indian family 474 residue is located in the C domain of the PK subunit that is essential for both intersubunit contact and allosteric regulation. As this enzyme shows catalytic activity only as a dimer or tetramer, it is rational that the structural alteration would result in severe PK deficiency (Kanno et al., 1994). PK deficiency hemolytic anemia, described in the Old Order Amish of Pennsylvania (Bowman et al., 1965), was much more severe than that reported by Tanaka et al. (1962). The mutation was subsequently characterized by Kanno et al. (1994). The affected individual in this report had severe hemolytic anemia which could have led to death in the first years of life if not treated by transfusions and splenectomy. In the family referred to us, the propositus was also homozygous for the 1436GA mutation. As we had already characterized the mutation, it allowed a rapid and specific prenatal diagnosis by MspI restriction analysis which was also confirmed by DNA sequencing. The fetus was found to be heterozygous and the pregnancy was continued. There are only a handful of reports on prenatal diagnosis of PK deficiency. There are two reported cases where prenatal diagnosis was done using amniotic fluid cells in one case and cord blood in the other by PCR and restriction enzyme analysis as well as linkage analysis using two polymorphic sites linked to the PKLR gene (Baronciani and Beutler, 1994). In 1996, prenatal diagnosis of PK-Monder due to a frame shift mutation in the PKLR gene causing severe hereditary non-spherocytic anemia was done in France (Rouger et al., 1999). In India, PK deficiency had remained an under-diagnosed disease. In the last five years we encountered 15 cases of PK deficiency (Colah and Kedar, 2006; Kedar et al., 2006), and hence, this erythro-enzymopathy is not that uncommon in India. Fetal tissue in our case was obtained by CVS to enable diagnosis in the first trimester of pregnancy. The possibility of phenotypic variation in PK deficiency should be borne in mind when counseling families at risk about the likely clinical effects in an affected child. 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