Available online at www.annclinlabsci.org Annals of Clinical & Laboratory Science, vol. 41, no. 2, 2011 197

A novel PHKA2 gross deletion mutation in a Korean patient with X-linked liver glycogenosis type I

Kyoung-Jin Park1, Hyung-Doo Park1, Soo-Youn Lee1, Chang-Seok Ki1, Yon-Ho Choe2 1Department of Laboratory Medicine & Genetics, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea; 2Department of Pediatrics, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea

Abstract. X-linked liver glycogenosis (XLG) is caused by a mutation in the PHKA2 gene which encodes the alpha subunit of phosphorylase kinase (PHK). Although XLG is not a rare disease, there have been no reports of PHKA2 mutations in Koreans. A 5-year-old boy presented with easy fatigability and hepatomegaly. Liver enzymes were increased and liver histology revealed deposition of glycogen. The PHK activity was markedly decreased compared to control. No amplification was observed at exon 8 of the PHKA2 gene, as a result of the deletion of exon 8. Sequence analysis revealed a hemizygous deletion in the region of exon 8 (c.717+781_864+225del1626). The patient was diagnosed as having XLG I. To the best of our knowledge, this is the first report of XLG I in Koreans.

Keywords: glycogen storage disease; Korean patient; phosphorylase kinase; X-linked liver glycogenosis

List of abbreviations: GSD, Glycogen storage disease; PCR, polymerase chain reaction; PHK, phosphorylase kinase; XLG, X-linked liver glycogenosis

Introduction hepatic glycogenosis (Figure 1). PHK enzyme has a hexadecameric structure comprised of Glycogen storage diseases (GSDs) are inherited subunits (αβγδ)4. Differences in these subunits metabolic disorders caused by deficiencies of various as well as their isoenzymes result in variations enzymes involved in the degradation or synthesis in mode of inheritance, tissue specificity, and of glycogen. GSD type IX, due to a deficiency clinical manifestations [1-4]. The most common of phosphorylase kinase (PHK, EC 2.7.11.19) subtype of PHK deficiency (75%) is X-linked comprises one quarter of all GSD diagnosed [1]. liver glycogenosis (XLG; MIM 306000) due to For glycolysis, glucagon activates the membrane mutations in the PHKA2 gene that encodes the bound adenylate cyclase and interacts with the alpha subunit of PHK [1-4]. regulatory subunits of the protein kinase, resulting in phosphorylation of PHK. This activated PHK XLG is divided into two subtypes with similar converts the glycogen phosphorylase into its active clinical features but different enzyme activity, form, which is defective in GSD IX, resulting in XLG I and XLG II. PHK enzyme deficiency is apparent in both liver and blood cells for XLG I, Address correspondence and reprint requests to Hyung-Doo Park, but only in the liver for XLG II [1-4]. According Department of Laboratory Medicine & Genetics, Samsung Medical to Hendrickx et al. [4-7], the different biochemical Center, Sungkyunkwan University School of Medicine, 50 Ilwon- phenotypes are due to the different nature of dong, Gangnam-gu, Seoul, 135-710, Korea; tel +82-2-3410-0290; fax +82-2-3410-2719; e-mail [email protected]; or to the mutations. XLG I mutations are caused by Yon-Ho Choe, Department of Pediatrics, Samsung Medical nonsense or frameshift mutations, whereas XLG Center, Sungkyunkwan University School of Medicine, Seoul, II mutations resulted from missense mutations or Korea; tel +82.2-3410-3527 ; fax +82.2-3410- 0043 ; e-mail small in-frame deletions or insertions. [email protected] 0091-7370/11/0200-197. © 2011 by the Association of Clinical Scientists, Inc. 198 Annals of Clinical & Laboratory Science, vol. 41, no. 2, 2011 percentile). His parents claimed that they were not consanguineous and had neither specific perinatal problems nor a family history of any specific disease. He had mild hepatomegaly, but no other specific symptoms were observed upon physical examination. Liver enzymes were increased, while others were within normal ranges on routine laboratory tests: aspartate aminotransferase 123 U/L (reference range, 0-40 U/L), alanine aminotransferase 112 U/L (reference range: 0-40 U/L). Serologic markers for viral hepatitis were all negative Fig 1. Metabolic pathway and clinical manifestations of and antinuclear antibodies associated with glycogen storage disease IX autoimmune hepatitis were not detected. The ceruloplasmin level was 18.2 mg/dL (reference range: 20-60 mg/dL), but the copper level in 24-hour urine was not increased (29.7 ug/day; reference range, 0-38 ug/day). Liver histology showed depositions of glycogen under electron microscopy, indicative of GSD (Figure 2).

Biochemical analysis The PHK activity was measured in erythrocytes according to a previously described method [8]. Erythrocytes, isolated following collection in heparin coated Fig. 2: Electron microscopy in the liver biopsy sample with tube, were diluted four-fold with distilled water. GSD IX shows glycogenation of the nuclei of the hepatocytes Hemoglobin (gHb) was determined using an and cytoplasmic accumulation of the glycogen particles. automated hematology analyzer (Beckman Coulter, Miami, FL). Samples (25 uL) were incubated with reagent mixture (25 uL) in XLG is characterized by mild growth Eppendorf tubes in duplicate. The reagent retardation, hepatomegaly, and elevation of mixture contained 90 U/mL of phosphorylase liver enzymes. Unlike other types of GSD, b, 20 mmol/L of magnesium acetate, 12 severe hypoglycemia and metabolic acidosis are mmol/L of ATP, and 80 mmol/L of Tris-β- rare in XLG. In addition, clinical symptoms glycerophosphate buffer (pH 6.8). Incubation or laboratory abnormalities improve with for 0 and 15 minutes at 37°C was completed and age. These benign features may be responsible 600 uL of stop solution containing 0.1 mol/L for under-diagnosis of the disease. Here, we of NaF and 5 mmol/L of EDTA was added to describe a Korean patient with XLG I confirmed 20 uL of the mixture. The PHK activity was by biochemical and genetic analysis. determined by calculating the difference in Materials and Methods phosphate amounts from two tubes incubated for 0 min (C0) and 15 min (C15). Case presentation A 5-year-old boy presented with easy fatigability and hepatomegaly Genetic analysis Genomic DNA was isolated since the age of two. He also had mild from peripheral blood using a Wizard® hypoglycemic symptom and frequent epistaxis. Genomic DNA Purification Kit (Promega, He was referred to our hospital for further Madison, WI, USA). Polymerase chain reaction evaluation of hepatomegaly and elevated liver (PCR) of whole exons of the PHKA2 gene enzymes. His height was 100.5 cm (3th - 10th was performed using a Model 9700 thermal percentile) and weight was 17.3 kg (10th - 25th X-linked liver glycogenosis type 1 in Korea 199 cycler (Applied Biosystems, Foster City, CA, USA). Direct sequencing was performed with the ABI prism 3100 Genetic analyzer (Applied Biosystems). The reference sequence used was NM_000292.2 from the National Center for Biotechnology Information Database. PHKA2 gene primers were designed in our institution (sequence available upon request). Long-range PCR was performed to confirm the deletion in the PHKA2 gene. Primer sets were: 5’- ttgcttaatgaaaaaggaacacc-3’ for exon Fig 4. Sequence analysis of the deletion of exon 8 in the PHKA2 7, 5’-tgacttctcgcctgaggaat-3’ for exon 8, and gene. Sequence analysis showed a deletion of 1,626 base pair 3’-ccagctcaccgtccctacta-5’ for exon 9. The from intron 7 to intron 8 of the PHKA2 gene, described as reaction conditions were 94°C for 30 sec, c.717+781_864+225del1626 58°C for 30 sec, and 72°C for 3 min during 35 exon 8, suggesting its deletion. Long-range cycles. The deletion endpoint was determined PCR confirmed the deletion by identifying using primer 3’-cgatagtttcaactggctgga-5’ a shorter fragment in the patient than in with sequences close to the deleted region by the normal controls (Figure 3). Deletion primer walking. This study was performed after endpoints were determined by sequencing obtaining informed consent from the patient’s analysis and those were c.717+781 at intron parents. 7 and c.864+225 at intron 8. This suggested Results that the patient was a hemizygote for the large deletion with 1,626 base pairs (bp) in PHKA2 (Figure 4). Other sequences in the PHKA2 The patient’s PHK activity in erythrocytes was gene were all identical to reference sequences. markedly decreased to 6.57 umol/min/gHb. According to the nomenclature from HGVS, Two healthy subjects showed PHK activity of the PHKA2 mutation may be described as 78.29 and 107.71 umol/min/gHb, respectively. c.717+781_864+225del1626. PCR of the PHKA2 gene failed to amplify Discussion

PHK is a complex enzyme made up of four different subunits (αβγδ)4. Mutations in the different subunits and their isoforms result in heterogeneous phenotypes. According to the mode of inheritance and tissue involvement, there may be six different subtypes (1-6): (1) GSD IXa indicating XLG, (2) GSD IXb showing combined liver and muscle PHK deficiency, (3) GSD IXc showing autosomal liver PHK deficiency, (4) GSD IXd showing X–linked muscle PHK deficiency, (5) GSD IXe showing autosomal muscle PHK deficiency, and (6) GSD IXf showing heart PHK deficiency. The alpha subunit of PHK is encoded Fig 3. Long-range polymerase chain reaction (PCR) analysis of by two different genes on the X chromosome, the PHKA2 gene. The fragments of lane 1-4 are from normal PHKA2 for liver isoform and PHKA1 for muscle controls and those of lane 5-6 from the patient. PCR fragment isoform. The beta subunit is encoded by the by 8F/9R primers was not amplified in lane 5. PCR product of 7F/9R primer set in lane 6 was shorter than those of the normal PHKB gene while the gamma subunit is encoded controls in lane 2 and 4. F, forward primer; R, reverse primer by the PHKG gene (PHKG2 gene for testis/liver 200 Annals of Clinical & Laboratory Science, vol. 41, no. 2, 2011 isoform and PHKG1 gene for muscle isoform). In summary, we report the first case of XLG I in PHK activity can be decreased by mutations in a Korean patient diagnosed using biochemical any of the subunits. Thus, genetic analysis may and genetic analysis. XLG is the mildest type be necessary for a definitive diagnosis of the GSD of GSD and asymptomatic with age, which IX subtype. suggests that many cases may go undiagnosed. Clinical suspicion, biochemical analysis, and Here we reported a case of XLG due to a mutation selective genetic analysis may be essential for a in the PHKA2 gene. The patient had growth definitive diagnosis of XLG. retardation for his age and mild hypoglycemic symptoms as well as mild hepatomegaly for three Acknowledgements years. We excluded viral hepatitis based on the results of viral serology tests. Also, the possibility This study was supported by a grant from the of Wilson disease was low based on the levels Korean Ministry of Education, Science and of ceruloplasmin and copper. Liver histology Technology, FPR08A2-130 of the 21C Frontier suggested the diagnosis of GSD. Mild clinical Functional Proteomics Program. manifestations suggested GSD IV or IX. These two GSD types are clinically indistinguishable. References Considering that the patient was male and most cases of GSD IX are inherited by the X-linked 1. Burwinkel B, Shin YS, Bakker HD, Deutsch J, Lozano MJ, Maire I, Kilimann MW. 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Variability of biochemical and clinical phenotype in X-linked liver glycogenosis with mutations in the phosphorylase kinase It is very interesting that both XLG I and XLG PHKA2 gene. Hum Genet 1998;102:423-429. II with different enzyme activity are caused by 4. Hendrickx J, Lee P, Keating JP, Carton D, Sardharwalla IB, Tuchman M, Baussan C, Willems PJ. Complete genomic a mutation in the same gene. PHK activity is structure and mutational spectrum of PHKA2 in patients with decreased in both peripheral blood cells and x-linked liver glycogenosis type I and II. Am J Hum Genet liver for XLG I, whereas normal in peripheral 1999;64:1541-1549. 5. Hendrickx J, Coucke P, Dams E, Lee P, Odievre M, Corbeel blood cells and decreased in liver for XLG II. L, Fernandes JF, Willems PJ. Mutations in the phosphorylase XLG I mutations might induce disruption of kinase gene PHKA2 are responsible for X-linked liver glycogen the protein and absence of the alpha subunit. storage disease. 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