Clinical and genetic characteristics of 17 Chinese patients with glycogen storage disease type IXa

Gene 627 (2017) 149–156

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Research paper

Clinical and genetic characteristics of 17 Chinese patients with glycogen storage disease type IXa

MARK

Jiangwei Zhanga,1, Yuheng Yuanb,1, Mingsheng Mab, Yan Liub, Weimin Zhangc, Fengxia Yaoc, Zhengqing Qiub,⁎ a b c

Department of Pediatrics, Peking University International Hospital, Beijing 102206, China Department of Pediatrics, Peking Union Medical College Hospital, Peking Union Medical College, Chinese Academy of Medical Sciences, Beijing 100730, China Genetics Research Laboratory, Peking Union Medical College Hospital, Peking Union Medical College, Chinese Academy of Medical Sciences, Beijing 100730, China

A R T I C L E I N F O

A B S T R A C T

Keywords: Gene mutation Glycogen storage disease type IX PHKA2 gene Phosphorylase kinase

Glycogen storage disease (GSD) type IXa is caused by PHKA2 mutation, which accounts for about 75% of all the GSD type IX cases. Here we first summarized the clinical data and analyzed the PHKA2 gene of 17 Chinese male patients suspected of having GSD type IXa. Clinical symptoms of our patients included hepatomegaly, growth retardation, and liver dysfunction. The clinical and biochemical manifestations improved and even disappeared with age. We detected 14 mutations in 17 patients, including 8 novel mutations; exons 2 and 4 were hot spots in this research. In conclusion, glycogen storage disease type IXa is a mild disorder with a favorable prognosis, and there was no relationship between genotype and phenotype of this disease.

1. Introduction Glycogen storage disease type IX (GSD type IX) is one of the most common types of glycogen storage diseases, accounting for approximately 25% of patients with GSD and caused by a deficiency of phosphorylase kinase (PhK) (Achouitar et al., 2011). Phosphorylase kinase (PhK, EC.2.7.1.38) is composed of four different subunits with four copies of each (αβγδ)4 and is responsible for catalyzing inactive phosphorylase into an active form, which initiates glycogen degradation. Each of the subunits has tissue-specific isoforms encoded by different genes or alternative splicing of a single gene (van den Berg et al., 1995; Beauchamp et al., 2007). Glycogen storage disease type IXa (MIM 306000), which is caused by mutations in the PHKA2 gene, formerly known as X-linked liver glycogenosis (XLG), results from the absence of liver-α subunit and accounts for about 75% of all the GSD type IX cases (Achouitar et al., 2011). The PHKA2 gene is located at Xp22.2–22.1 and contains 33 exons (Davidson et al., 1992). According to the Human Gene Mutation Database (HGMD, http://www.hgmd.cf.ac.uk/), 99 PHKA2 mutations have been reported, including missense/nonsense mutations, splicing, insertion, and deletion mutations. Clinical symptoms of type IXa

include growth retardation and hepatomegaly. Biochemical abnormalities include elevated liver transaminase, hypercholesterolemia, hypertriglyceridemia, hypoglycemia, and occasional elevated levels of uric acid and lactic acid (Hirono et al., 1998). These clinical and biochemical manifestations improve and even disappear with age. In addition, adult height of the patients tends to be normal (Schippers et al., 2003). Approximately 134 cases have been detected and included 5 Chinese patients who came from Hong Kong and Taiwan (Chen et al., 2009; Lau et al., 2011). Here we report 17 Chinese patients with glycogen storage disease type IXa in whom the condition was diagnosed by sequencing analysis. 2. Materials and methods 2.1. Patients There were 17 male patients suspected of GSD type IX, with age of onset between 3 months and 10 years. Age of last evaluation was 2 to 28 years old, and the patients were referred to the Department of Pediatrics of Peking Union Medical College Hospital. All patients showed abdominal distension, increased liver

Abbreviations: GSD, glycogen storage disease; PhK, phosphorylase kinase; XLG, X-linked liver glycogenosis; ALT, aminotransferase; AST, aspartate aminotransferase; CR, serum creatinine; TC, total cholesterol; TG, total triglyceride; CK, creatine kinase; NGS, next-generation sequencing; PCR, polymerase chain reaction; PAS, periodic acid-Schiff; cAMP, cyclic adenosine monophosphate; PKA, dependent protein kinase ⁎ Corresponding author at: No. 53, Dongdan North Street, Dongcheng District, Beijing 100730, China. E-mail addresses: [email protected] (M. Ma), [email protected] (Z. Qiu). 1 These authors contributed equally to this work and share first authorship. http://dx.doi.org/10.1016/j.gene.2017.06.026 Received 12 February 2017; Received in revised form 4 May 2017; Accepted 12 June 2017 Available online 13 June 2017 0378-1119/ © 2017 Published by Elsevier B.V.

150 c.392G > A p.Gly131Asp

p.Arg295His

Hepatomegaly Growth retardation 114 (< p3) 120 (< p3) 21.5 (p3–10) 23 (< p3) 9 4.5 77 20 44 22 5.1 4.8 4.43 4.85 0.82 1.23 1.06 NR NR NR PAS(+) Fibrosis Necrosis Steatosis c.2726_2727 delTT

0.3 9 10 10.3

3

Liver size is below the right costal margin detected by ultrasonic. ALT, alanine aminotransferase, AST, aspartate aminotransferase, TC, total cholesterol, TG, triglyceride. T1, age of first visit to hospital, T2, age of the last follow-up, T3, age of stopping cornstarch. NR, not recorded.

PHKA2 gene mutations

Liver biopsy

Uric acid (N: 150–357 μmol/L)

Lactic acid (N: 0.5–1.6 mol/L)

TG (N: 0.45–1.70 nmol/L)

TC (N: 2.85–5.70 nmol/L)

Glucose (N: 3.9–6.1 mmol/L)

AST (N: 5–37 U/L)

ALT (N: 5–40 U/L)

Liver size (cm)

124 (p10–25) 174 (p25–50) 25 (p25–50) 61 (p50–75) 2.5 (–) 149 20 154 30 3.9 5.3 4.13 NR 0.75 1.16 NR NR NR 568 PAS(+) Necrosis Steatosis

6 8 17 17 11.3 Increased transaminase

2

c.884G > A

81 (< p3) 172 (p25–50) 14 (p75–90) 62 (p50–75) 7.6 3.2 37 35 25 20 4 4.3 3.12 3.23 1.32 1.51 NR 1.36 NR 317

Height (cm)

Weight (kg)

0.5 2.3 28 26 18 Hepatomegaly anemia

Age of onset (years) T1 (years) Age of diagnosed (years) T2 (years) T3 (years) Chief complaint

T1 T2 T1 T2 T1 T2 T1 T2 T1 T2 T1 T2 T1 T2 T1 T2 T1 T2 T1 T2

1

Patient

Table 1 Summary of the clinical data of the 17 patients with GSD type IXa (patients 1–9).

p.Arg295His

c.889G > A

85 (< p3) 174 (p50–75) 14 (p50–75) 90 (> p97) 9 4.5 335 40 258 24 5 3.9 8.87 3.92 2.28 1.51 NR NR NR NR PAS(+) Necrosis

1 2.5 23 19 14 Hepatomegaly

4

p.Arg916Trp

c.2746C > T

Hepatomegaly Increased transaminase 105.5 (p25–50) 134.1 (p75–90) 19 (p50–75) 28 (p50–75) 3.9 3.1 88 18 75 26 3.53 4 3.54 4.24 0.54 0.5 0.7 NR 321 NR PAS(+) Fibrosis Necrosis

3 4.5 9 8

5

p.His113Arg

c.338A > G

2 2.3 15 14.3 13.5 Hepatomegaly Frequent hunger 87 (p25–50) 168.5 (p50–75) 12.5 (p25–50) 67 (p75–90) 10 2 495 25 457 32 5.6 4.9 4.12 3.56 1.41 0.98 NR NR NR NR PAS(+) Fibrosis

6

p.Arg45Trp

c.133C > T

89.5 (< p3) 113.5 (< p3) 13.5 (p10–25) 21 (p10–25) 10 3.5 579 124 683 104 3 3.9 5.12 4.53 0.87 1.12 2.5 NR 325 288

Hepatomegaly

3 3 7 7

7

c.237 + 1 G > T

c.237 + 1 G > T

2 2 9 9 7 Hepatomegaly Frequent hunger 80 (< p3) 120.5 (< p3) 10 (< p3) 21 (< p3) 4.7 4.1 1014 37 649 47 5.2 5 6.18 3.43 3.68 0.98 1.8 NR NR 200 PAS(+) Fibrosis Necrosis

8

p.Arg916Trp

c.2746C > T

Hepatomegaly Increased transaminase 74 (p10–25) 121 (p25–50) 11 (p75–90) 32 (p90–97) 9.5 (−) 527 18 375 27 3.1 4.8 9.42 5.06 8.78 1.45 NR NR NR 177

0.7 1 7.8 7.3

9

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Table 2 Summary of the clinical data of the 17 patients with GSD type IXa (patients 10–17). Patient

10

11

12

13

14

15

16

17

Age of onset (years) T1 (years) Age at diagnosis (years) T2 (years) T3 (years) Chief complaint

1 2.5 2.5 2.5 – Hepatomegaly Increased transaminase

1 2.5 6.6 6.6 – Hepatomegaly Increased transaminase Frequent hunger Fatigue

0.8 1.5 1.5 2.1 – Hepatomegaly Increased transaminase Frequent hunger

0.8 1.8 2 2 – Hepatomegaly Fatigue

10.5 10.5 11 11 – Increased transaminase

2 3.1 3.8 3.6 – Hepatomegaly Increased transaminase

5 7.1 7.1 7.1 – Increased transaminase

1.8 2.3 8.9 8.9 – Hepatomegaly

Growth retardation

Frequent hunger

Growth retardation 80 (p10)

82 (p10–25) 12 (p50–75)

4.4

4.3

711

153

488

226

1.8

3.44

3.34

4.67

96.5 (p25–50) 102 (p50) 16 (p75–90) 18 (p50–75) 7 6.8 181 45 206 49 3.2 3.2 4.75 5.56

102 (< p3)

12.5 (p50–75)

132 (p10) 139.5 (p10–25) 25 (p3–10) 34 (p50) 0 1.9 106 31 126 33 3.1 4.2 5.59 5.81

81 (< p3) 122 (< p3) 13 (p25–50) 23 (p3–10) 7.5 2.7 527 295 1101 177 2.4 4.4 4.57 5.26

1.92

1.14

0.93 1.83

1.69 1.18

2.7

1.13 0.88

2.5

1.38

1.4

363.5

0.92 0.99 279 272

4.26

216

1.03 1.36 342 251

487

274 253

PAS(+) Fibrosis Necrosis c.884G > A p.Arg295His

c.134G > A p.Arg45Gln

c.538G > A p.Asp180Asn

c.884G > A p.Arg295His

Height (cm) Weight (kg) Liver size (cm) ALT (N: 5–40 U/L) AST (N: 5–37 U/L) Glucose (N: 3.9–6.1 mmol/L) TC (N: 2.85–5.70 nmol/ L) TG (N: 0.45–1.70 nmol/ L) Lactic acid (N: 0.5–1.6 mol/L) Uric acid (N: 150–357 μmol/ L) Liver biopsy

PHKA2 gene mutations

T1 T2 T1 T2 T1 T2 T1 T2 T1 T2 T1 T2 T1 T2 T1 T2

88 (p3–10) 13.5 (p25–50) 3 625 85 4.1 4.76

0.92

T1 T2 T1 T2

88 (p3–10) 118 (p50) 13 (p25–50) 22 (p50) 3.5 2 682 22 952 26 4.9 4.8 3.87 5.08 1.24 1.07

0.91 247 PAS(+) Fibrosis c.1498C > T de novel

c.407A > T p.Asp136Val

c.3377C > A p.Ser1126Ter

c.1925C > G p.Ser642Ter

18 (< p3) 0 133 136 5.4 5.12

Liver size is below the right costal margin detected by ultrasonic. ALT, alanine aminotransferase, AST, aspartate aminotransferase, TC, total cholesterol, TG, triglyceride. T1, age of first visit to hospital, T2, age of the last follow-up, T3, age of stopping cornstarch. NR, not recorded.

2.3. Bioinformatics analysis

transaminase, growth retardation, and fasting hypoglycemia or hyperlipidemia at the first visit. Fasting blood glucose, blood alanine aminotransferase, aspartate aminotransferase, serum creatinine, urea, total cholesterol (TC), triglyceride (TG), creatine kinase, and liver ultrasound were assessed during every hospital visit. Informed consent was obtained from the subjects' parents.

High-quality reads were identified by filtering out low-quality reads and adaptor sequences using Solexa QA package and Cutadapt program, respectively. Variants were first selected if they appeared in the 1000 Genomes Project database with an MAF > 0.05. The remaining variants were further processed according to the Single Nucleotide Polymorphism Database (dbSNP) database. SNPs and indels were identified using SOAP snp and GATK programs. Subsequently, the reads were realigned to the reference genome (NCBI37/hg19) using BWA software.

2.2. Targeted region capture sequencing library preparation Genomic DNA was extracted from peripheral ethylenediaminetetraacetic acid-treated blood using Blood Genomic Extraction Kit (Qiagen, Germany), and quantified using a NanoDrop 2000 unit (Thermo Fisher Scientific, Wilmington, DE). Next-generation sequencing with exome sequencing with GSD panels was performed. GSD panels included GYS1, GYS2, G6PC, SLC37A4, GAA, AGL, GBE1, PYGM, PFKM, PYGL, PHKA1, PHKA2, PHKB, PRKAG2, PHKG2, PGAM2, LDHA, ALDOA, ENO3, PGM1, GYG1 and LAMP2. A minimum of 3 μg of DNA was used to prepare indexed Illumina libraries according to the manufacturer's protocol. The final library size was 300 to 400 bp, including the adapter sequences. The DNA of the targeted region was captured from the genomic DNA library using the Agilent SureSelect kit. The enriched libraries were sequenced on an Illumina Solexa HiSeq 2000 sequencer for paired-end reads of 100 bp.

2.4. Validation by Sanger sequencing The gene sequence was checked from UCSC-Genomes (NM_000292). Forward and reverse primers of every exon were designed by Primer3Web software. Annealing temperature of each reaction was selected using agarose gel electrophoresis and image analysis. PHKA2 gene sequencing was performed by polymerase chain reaction (PCR), and the reaction mixture consisted of 12.5 μL PCR Mix (2 × EasyTap PCR Super Mix, TransGen, China), 10.5 μL distilled deionized H2O, 1 μL DNA, 0.5 μL forward primer, and 0.5 μL reverse primer. Each PCR amplification was performed for 35 cycles of 95 °C for 5 min, 94 °C for 30 s, and 72 °C for 45 s, and then with a different 151

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c.133C>T, p.Arg45Trp

c.134G>A, p.Arg45Gln (reverse)

c.237 +1 G>T

c.338A>G, p.His113Arg

c.392G>A, p.Gly131Asp

c.407A>T, p.Asp136Val

c.538G>A, p.Asp180Asn (reverse)

c.884G>A, p.Arg295His

c.899G>A, p.Gly300Asp

c.1498C>T, p.Arg500Ter

Fig. 1. PHKA2 sequence of 17 patients in this research.

of onset ranged from 3 months to 10 years and the median age was 1.8 years. Age of first visit to the hospital ranged from 1 year to 10 years, and the median age was 2.5 years. Age of diagnosis ranged from 1.5 years to 28 years, and the median age was 8.9 years. All patients were treated with uncooked cornstarch (1.0 to 1.6 g/kg) four times a day. Five of them (patients 1, 2, 4, 6, 8) stopped taking cornstarch at the age of 7, 11, 13.5, 14, and 18 years. Patient 17 began taking cornstarch once every night at age 7 years.

annealing temperature for 10 min. PCR products were sequenced in a biotechnology company (Beijing Ruibo Xingke Biotechnology Company). Sequencing was compared using Chromas software and NCBI-BlastNucleotide blast. The protein conservation of novel missense mutations was predicted by HomoloGene software and the pathogenicity was predicted by SIFT and PolyPhen-2 software.

3. Results 3.1. Initial presentation The 17 patients came from 17 unrelated families of unconsanguineous couples. Clinical and biological information at the time of diagnosis and the last evaluation are detailed in Tables 1 and 2. Age

The chief complaints included abdominal distension (14 patients), increased liver transaminase level (9 patients), growth retardation (3 152

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J. Zhang et al. Fig. 1. (continued)

c.1925C>G, p.Ser642Ter (reverse)

c.2726_2727delTT, p.Phe909Cysfs31x

c.2746C>T, p.Arg916Trp(reverse)

c.3377C>A, p.Ser1126Ter (reverse)

(23%). Blood lipid levels were normal in all patients. Lactic acid levels were normal in all four tested patients. None of the patients developed liver adenoma.

patients), frequent hunger (1 patient), and hepatomegaly with anemia (1 patient). Physical examination showed short stature in 7 patients (41%) and liver enlargement in 15 patients (88%). None of the patients had intellectual disability. Biochemical tests showed fasting hypoglycemia in 8 patients (47%). Patient 16 had been treated at a local hospital and therefore showed both normal liver size and normal glycemia. Increased liver transaminase was present in 16 patients except for patient 1, who had been treated at a local hospital. Total cholesterol and triglyceride were elevated in 3 patients (17%). Mild elevation of lactic acid was detected in 5 of 10 tested patients (50%). Levels of serum creatinine and urea were normal in all patients. Eight of our patients underwent liver biopsy before treatment. All patients showed glycogen storage as confirmed by periodic acid-Schiff staining and inflammation on gross pathology. Fibrosis, necrosis, and steatosis were seen in six, six, and two patients, respectively. The PhK-activity in erythrocytes could not be determined due to technical constraints.

Table 3 Results of PHKA2 mutations of the 17 patients with GSD type IXa. SIFT

PolyPhen

6% 6% 6% 6%

Damaging

Probably damaging

4 4

6% 6%

Damaging

p.Asp180Asn

6

6%

Damaging

Probably damaging Probably damaging

p.Arg295His p.Gly300Asp

9 9

17% 6%

Damaging

p.Arg500Ter p.Ser642X p.Phe909Cys fs31x p.Arg916Trp p.Ser1126X

15 18 25

6% 6% 6%

25 32

11% 6%

Base change

Amino acid change

Exon

Frequency in this research

c.133C > T c.134G > A c.237 +1 G > Ta c.338A > Ga

p.Arg45Trp p.Arg45Gln p.His113Arg

2 2 2 4

c.392G > A c.407A > Ta

p.Gly131Asp p.Asp136Val

a

c.884G > A c.889G > Aa

c.538G > A

3.2. Long-term outcome Age of last evaluation ranged from 2 to 28 years and the median age was 7 years. Thirteen patients were followed up for 0.5 to 25.7 years. Patients 10, 12, 13, and 16 received a recent diagnosis and there were no follow-up data. In the last evaluation of 13 patients, 9 had normal height and 4 exhibited growth retardation. Hepatomegaly was still evident in 11 patients (84%) and fasting glucose levels were normal in 12 (92%). Liver transaminases were normal in 10 (77%) and slightly elevated in 3

a

c.1498C > T c.1925C > Ga c.2726_2727delTTa c.2746C > T c.3377C > A a

153

8 novel.

Possibly damaging

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Fig. 2. Conservation of amino acids that are changed by four novel missense mutations of the PHKA2 gene. The four changed residues are conserved in Homo sapiens (NP_000283.1), Pan troglodytes (XP_003317431.1), Macaca mulatta (XP_001084454.1), Canis lupus (XP_005641224.1), Bos taurus (NP_001178474.1), and Mus musculus (NP_001171350.1) et al.

p.His113Arg

p.Asp136Val

p.Asp180Asn

p.Gly300Asp

154

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Schippers HM et al. reported that patients with all 4 subtypes of GSD IX have a specific growth pattern characterized by initial growth retardation from age 2 to 10 years, a late growth spurt, and then complete catch-up with normal final height (Schippers et al., 2003). Sixteen patients in our research were younger than 10 years at the first visit; 7 of them (44%) exhibited growth retardation. In the last evaluation of 13 patients, 5 patients (including 2 adult patients) older than 10 years reached normal height. One patient who was slightly older than 10 years still showed growth retardation. The remaining 3 patients (43%) younger than 10 years still presented short stature. Four of the seven patients (57%) younger than 10 years manifested normal height before and after treatment, which was not completely consistent with the literature. This may be because these patients were not subdivided in the previous report or ethnic differences were noted in growth pattern of GSD type IXa. With regard to treatment, Schippers HM et al. proposed that GSD type IXa was a mild disorder requiring a mild diet, and it is questionable whether strict therapy should be used to obtain a normal growth pattern. Roscher et al. also reported that the majority of the patients who were not treated had normal liver enzyme levels and were of normal stature (Schippers et al., 2003; Roscher et al., 2014). Five of our patients stopped taking cornstarch at the age of 7 to 18 years old because of satisfactory biochemical results. After 1 to 10 years' follow-up, all 5 patients had normal laboratory data, 4 reached normal height, and 1 patient younger than 10 years old was still of short stature, which is in agreement with previous reports. Because our sample size was limited, the question whether cornstarch was essential for older patients with GSD type IXa remained for further research. Liver biopsy was rarely reported in patients with GSD IXa. Chronic liver fibrosis was a consistent finding in 3 patients; other gross pathology included variable steatosis, glycogen accumulation, inflammation, and no necrosis or cholestasis (Johnson et al., 2012; Tsilianidis et al., 2013; Roscher et al., 2014). Liver biopsies were performed in eight patients; necrosis was seen in six patients. Despite the necrosis, pathology revealed that clinical presentations and biochemical tests were similar to those of other patients. To understand the pathophysiology of necrosis in these patients, it is better to repeat the liver biopsy after treatment. Cho et al. reported a female Chinese patient carried a diseasecausing mutation of c.3614C > T manifesting mild hepatomegaly at the age of 29 years. Her medical history showed hepatomegaly and increased liver transaminase levels at 9 months old, which had been gradually improved. Explanation of this phenomenon was the nonrandom inactivation of normal X chromosome (Cho et al., 2013). Seventeen patients in our report were males and the 16 carrier mothers were all asymptomatic. PHKA2 gene encodes the liver-α subunit locates in Xp22.2–22.1 and contains 33 exons (Davidson et al., 1992). Thus far, approximately 134 patients were reported and 99 mutations have been recorded by HGMD, including 47 missense mutations, 28 deletion mutations, 9 insertion, 9 nonsense, and 6 splicing mutations. Currently only three mutations have been confirmed in Chinese patients from Hong Kong and Taiwan (Chen et al., 2009; Lau et al., 2011). In this report, we detected 14 mutations including 9 missense mutations, 3 nonsense mutation, 1 deletion mutation, and 1 splicing mutation; 8 mutations were novel. We did not identify the three previous reported Chinese mutations (c.136delG, c.3614 C > T, c.3111-1A > G) in our patients. The 14 mutations occurred in 8 of 33 PHKA2 exons (exons 2, 4, 6, 9, 15, 18, 25, and 32) and their introns, and 6 mutations (43%) gathered on exons 2 and 4, indicating that exons 2 and 4 were the hot spots in mainland Chinese patients. Four of eight novel mutations in our study were disease-causing mutations. The splicing mutation c.237 +1 G > T resulted in error splicing acceptor and may lead to skipping of the nearby exons. The nonsense mutation c.1498C > T led to the replacement of arginine with a termination codon at amino acid 500 (p.Arg500Ter), and the

3.3. PHKA2 analysis After PHKA2 gene sequencing, we found 14 mutations in 17 patients, including 9 missense mutations (c.133C > T, p.Arg45Trp; c.134G > A, p.Arg45Gln; c.338A > G, p.His113Arg; c.392G > A, p.Gly131Asp; c.407A > T, p.Asp136Val; c.538G > A, p.Asp180Asn; c.884G > A, p.Arg295His; c.889G > A, p.Gly300Asp; c.2746C > T, p.Arg916Trp), 3 nonsense mutations (c.1498C > T, p.Arg500X; c.1925C > G, p.Ser642X; c.3377C > A, p.Ser1126X), 1 deletion mutation (c.2726_2727delTT, p.Phe909Cys fs31x), and 1 splicing mutation (c.237 +1 G > T). Eight mutations of c.237 +1 G > T, c.338A > G, c.407A > T, c.538G > A, c.889G > A, c.1498C > T, c.1925C > G, and c.2726_2727delTT were novel (Fig. 1). All mothers were asymptomatic, and 16 of them were carriers of the mutations. The protein conservation and the pathogenicity of four novel missense mutations were studied by HomoloGene, SIFT, and PolyPhen-2 software (Table 3 and Fig. 2). 4. Discussion GSD type IX results from deficiency of PhK and the incidence is 1:100,000. PhK (E.2.7.1.38) is an initiator of glycogen degradation; it catalyzes inactive phosphorylase into the active form and plays an important role in regulating blood glucose. PhK is composed of four different subunits (αβγδ) with four copies of each. The subunits have tissue-specific isoforms coding by different genes except isoforms of the β-subunit that are formed by splicing of a single gene (van den Berg et al., 1995; Beauchamp et al., 2007). α, β, and δ subunits have regulatory function and inhibit the phosphotransferase activity of the γsubunit. Phosphorylation of the α and β subunits by the 3′, 5′-cyclic adenosine monophosphate–dependent protein kinase relieves inhibition of the γ-subunit and thereby activates PhK. Ca2 + also relieves inhibition through the δ subunit (Brushia and Walsh, 1999; Beauchamp et al., 2007). GSD IX is subdivided into four subtypes according to different encoding genes. Mutations of the PHKA2 gene, which resides on the X chromosome and encodes the liver-α subunit, leads to type IXa (Burwinkel et al., 1998). Approximately 134 patients with GSD IXa have been reported thus far (Ban et al., 2003; Roscher et al., 2014). GSD type IXa is formerly known as XLG (Achouitar et al., 2011). Growth retardation, hepatomegaly, elevated liver transaminase, hypercholesterolemia, hypertriglyceridemia, and mild hypoglycemia can be present. Levels of uric acid and lactic acid are usually normal (Hirono et al., 1998). These clinical and biochemical manifestations ameliorate and even disappear with age. Liver cirrhosis and hepatic adenoma have been deemed rare (Roscher et al., 2014). Currently, there are no reports regarding liver failure in patients with type IXa. In agreement with previous reports, 88% of our patients had hepatomegaly, 94% showed increased liver transaminase, 41% manifested growth retardation, 17% had elevated levels of blood lipids, 47% demonstrated fasting hypoglycemia, and 50% presented with slightly elevated lactic acid levels at presentation. At the last evaluation, 92% of patients had normal fasting glucose and 100% had normal blood lipid levels, 77% showed normal liver transaminase levels, 16% had normal liver size, and 100% had normal lactic acid levels. Patient 1 once achieved normal liver transaminase levels at the age of 20 years, but these levels increased without apparent inducement at the age of 25 years (alanine aminotransferase 146 U/L); liver transaminase levels normalized again at age 26 years. Hepatomegaly and increased liver transaminase were the most common clinical characteristics of patients with GSD type IXa. Fasting hypoglycemia was neither a significant nor persistent feature compared with GSD types Ia and III. There were no records of bleeding tendency, kidney dysfunction, or repeated infection in our patients. Our data further confirmed that the clinical symptoms of XLG were milder than other types of liver glycogen storage diseases [11] , and the prognosis of GSD type IXa was favorable. 155

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References

nonsense mutation c.1925C > G led to the replacement of serine to termination codon at amino acids 642 (p.Ser642X). The frameshift deletion mutation c.2726_2727 del TT led to the replacement of phenylalanine with cysteine at amino acid 909 and a premature presentation of termination codon at amino acid 940 (p.Phe909Cys fs31x). Four of eight novel missense mutations were not listed in the dpSNP database, and were further studied using HomoloGene, SIFT, and PolyPhen-2 software. Mutations of p.His113Arg, p.Asp136Val, p.Asp180Asn, and p.Gly300Asp were conserved in 20 species from human to Mus musculus, and predicted to be pathogenic. Therefore, considering the clinical manifestations of the patients, we predicted that these missense mutations were disease-causing mutations. There was no definite conclusion about the correlation of genotype and phenotype. Theoretically the deletion and splicing mutations should cause severe phenotype changes. A Japanese child with a large deletion spanning introns 19–26 and a Korean patient with gross deletion mutation were reported in 2007 and 2011. Their symptoms included hepatomegaly and liver dysfunction, and mild hypoglycemia was found only in the Korean child (Fukao et al., 2007; Park et al., 2011). Considering the symptoms of patients with nonsense, deletion, and splicing mutations in this research, we can draw the same conclusion that there was no definite relationship between genotype and phenotype of GSD type IXa. In conclusion, the present study is the first to investigate the clinical and genetic characteristics of 17 patients with GSD type IXa in mainland China. We detected 14 mutations, including 8 novel mutations; exons 2 and 4 were hot spots in this research. Our results further confirmed that GSD IXa is a mild disorder with a favorable prognosis. Hepatomegaly was the most persistent clinical sign. Fasting hypoglycemia was not significant and normal blood glucose control was achieved without cornstarch treatment in some teenage patients. There is no correlation between the phenotype and genotype.

Achouitar, S., Goldstein, J.L., Mohamed, M., Austin, S., Boyette, K., Blanpain, F.M., Rehder, C.W., Kishnani, P.S., Wortmann, S.B., den Heijer, M., Lefeber, D.J., Wevers, R.A., Bali, D.S., Morava, E., 2011. Common mutation in the PHKA2 gene with variable phenotype in patients with liver phosphorylase b kinase deficiency. Mol. Genet. Metab. 104, 691–694. Ban, K., Sugiyama, K., Goto, K., Mizutani, F., Togari, H., 2003. Detection of PHKA2 gene mutation in four Japanese patients with hepatic phosphorylase kinase deficiency. Tohoku J. Exp. Med. 200, 47–53. Beauchamp, N.J., Dalton, A., Ramaswami, U., Niinikoski, H., Mention, K., Kenny, P., Kolho, K.L., Raiman, J., Walter, J., Treacy, E., Tanner, S., Sharrard, M., 2007. Glycogen storage disease type IX: high variability in clinical phenotype. Mol. Genet. Metab. 92, 88–99. van den Berg, I.E., van Beurden, E.A., Malingre, H.E., van Amstel, H.K., Poll-The, B.T., Smeitink, J.A., Lamers, W.H., Berger, R., 1995. X-linked liver phosphorylase kinase deficiency is associated with mutations in the human liver phosphorylase kinase alpha subunit. Am. J. Hum. Genet. 56, 381–387. Brushia, R.J., Walsh, D.A., 1999. Phosphorylase kinase: the complexity of its regulation is reflected in the complexity of its structure. Front. Biosci. 4, D618–D641. Burwinkel, B., Amat, L., Gray, R.G., Matsuo, N., Muroya, K., Narisawa, K., Sokol, R.J., Vilaseca, M.A., Kilimann, M.W., 1998. Variability of biochemical and clinical phenotype in X-linked liver glycogenosis with mutations in the phosphorylase kinase PHKA2 gene. Hum. Genet. 102, 423–429. Chen, S.T., Chen, H.L., Ni, Y.H., Chien, Y.H., Jeng, Y.M., Chang, M.H., Hwu, W.L., 2009. X-linked liver glycogenosis in a Taiwanese family: transmission from undiagnosed males. Pediatr Neonatol 50, 230–233. Cho, S.Y., Lam, C.W., Tong, S.F., Siu, W.K., 2013. X-linked glycogen storage disease IXa manifested in a female carrier due to skewed X chromosome inactivation. Clin. Chim. Acta 426, 75–78. Davidson, J.J., Ozcelik, T., Hamacher, C., Willems, P.J., Francke, U., Kilimann, M.W., 1992. cDNA cloning of a liver isoform of the phosphorylase kinase alpha subunit and mapping of the gene to Xp22.2–p22.1, the region of human X-linked liver glycogenosis. Proc. Natl. Acad. Sci. U. S. A. 89, 2096–2100. Fukao, T., Zhang, G., Aoki, Y., Arai, T., Teramoto, T., Kaneko, H., Sugie, H., Kondo, N., 2007. Identification of Alu-mediated, large deletion-spanning introns 19–26 in PHKA2 in a patient with X-linked liver glycogenosis (hepatic phosphorylase kinase deficiency). Mol. Genet. Metab. 92, 179–182. Hirono, H., Shoji, Y., Takahashi, T., Sato, W., Takeda, E., Nishijo, T., Kuroda, Y., Nishigaki, T., Inui, K., Takada, G., 1998. Mutational analyses in four Japanese families with X-linked liver phosphorylase kinase deficiency type 1. J. Inherit. Metab. Dis. 21, 846–852. Johnson, A.O., Goldstein, J.L., Bali, D., 2012. Glycogen storage disease type IX: novel PHKA2 missense mutation and cirrhosis. J. Pediatr. Gastroenterol. Nutr. 55, 90–92. Lau, C.K., Hui, J., Fong, F.N., To, K.F., Fok, T.F., Tang, N.L., Tsui, S.K., 2011. Novel mutations in PHKA2 gene in glycogen storage disease type IX patients from Hong Kong, China. Mol. Genet. Metab. 102, 222–225. Park, K.J., Park, H.D., Lee, S.Y., Ki, C.S., Choe, Y.H., 2011. A novel PHKA2 gross deletion mutation in a Korean patient with X-linked liver glycogenosis type I. Ann. Clin. Lab. Sci. 41, 197–200. Roscher, A., Patel, J., Hewson, S., Nagy, L., Feigenbaum, A., Kronick, J., Raiman, J., Schulze, A., Siriwardena, K., Mercimek-Mahmutoglu, S., 2014. The natural history of glycogen storage disease types VI and IX: long-term outcome from the largest metabolic center in Canada. Mol. Genet. Metab. 113, 171–176. Schippers, H.M., Smit, G.P., Rake, J.P., Visser, G., 2003. Characteristic growth pattern in male X-linked phosphorylase-b kinase deficiency (GSD IX). J. Inherit. Metab. Dis. 26, 43–47. Tsilianidis, L.A., Fiske, L.M., Siegel, S., Lumpkin, C., Hoyt, K., Wasserstein, M., Weinstein, D.A., 2013. Aggressive therapy improves cirrhosis in glycogen storage disease type IX. Mol. Genet. Metab. 109, 179–182.

Acknowledgments This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. The authors declare no conflicts of interest. Yuheng Yuan and Jiangwei Zhang analyzed all the clinical and experimental data of these patients. Mingsheng Ma and Yan Liu collected the clinical data. Fengxia Yao and Weimin Zhang completed the gene analysis of these patients. We are very grateful to The National Key Research and Development Program of China (2016YFC0905100) for funding this research. We would like to thank the patients and their families for participating in the research.

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