Glycogen storage disease type IX (GSD IX) is caused by a general deficiency in phosphorylase kinase (PhK), which plays a major role in regulating the breakdown of glycogen. The PhK enzyme is composed of four copies each of four subunits (α, β, γ, and δ [also known as calmodulin]), which are encoded by PHKA1 and PHKA2 for the α subunits, PHKB for the β subunits, PHKG1 and PHKG2 for the γ subunits, and CALM1, CALM2, and CALM3 for the δ subunits [1]. There are autosomal recessive forms of GSD IX (caused by mutations in PHKB, PHKG1, PHKG2, CALM1, CALM2, and CALM3), a X-linked liver form (caused by mutations in PHKA2), and a X-linked muscle form (caused by mutations in PHKA1). Specifically, the α subunits, which are encoded by the PHKA2 gene in liver and by PHKA1 in muscle, regulate the activity of the catalytic γ subunits, which carry out the function of PhK. Mutations in PHKA2 are the most common cause of PhK deficiency [2].

The clinical manifestation of GSD IX stemming from PHKA2 mutation is characterized by childhood onset of hepatomegaly, growth retardation, fasting ketosis, and fasting hypoglycemia [3]. Symptoms and biochemical abnormalities in GSD IX patients have been reported to improve with age [4]. However, several GSD IX patients possessing PKHA2 mutations have been reported to progress to liver cirrhosis, or even present with cirrhosis at the time of diagnosis [4, 5].

GSD is a clinically and genetically heterogeneous group of diseases. Different types of GSD can sometimes be clinically indistinguishable; for example, both GSD VI and GSD IX often manifest with hepatomegaly with short stature [3]. Molecular diagnosis of GSD provides the advantage of avoiding invasive liver biopsy [6] and is an important starting point for appropriate therapeutic and monitoring plans [7]. Furthermore, molecular diagnosis allows determination of the inheritance pattern (autosomal recessive vs. X-linked) and DNA testing for other family members when the mutation in the proband is known.

The PHKA2 gene is located at chromosomal locus Xp22.2-p22.1 and consists of 33 exons spanning more than 65 kb and encoding a protein of 1,235 amino acids [8]. Sequence analysis of this X-linked gene can detect small intragenic deletions/insertions and missense, nonsense, and splice site mutations in males and females, and lack of amplification by PCR prior to sequence analysis can suggest a putative exonic or whole-gene deletion on the X chromosome in affected males [9]. Understanding the mutation spectrum of causative genes in different ethnic groups could provide background knowledge to develop a tailored diagnostic approach for different patient populations [7].

To date, data on PHKA2 mutations in Korean patients with GSD IX are available for only two cases. Thus, the aim of this study was to evaluate the mutation spectrum in Korean patients with GSD IX and compare it with previously reported mutation spectra in other ethnic populations.

In this study we examined the PHKA2 mutation spectrum in Korean patients with GSD IX. GSD IX is associated with a deficiency in liver PhK (caused by mutations in PHKA2, PHKB and PHKG2) or the muscle form of PhK (caused by mutations in PHKA1). Mutations in PHKA2 have been reported to be the most common cause of GSD IX (responsible for approximately 75 % of cases) [20, 21]. The second most common cause of liver PhK deficiency is mutations in PHKG2 (autosomal recessive), followed by PHKB (also autosomal recessive) [3]. X-linked liver glycogenosis (XLG) caused by PHKA2 mutations can be divided into two subtypes: XLG I with no detectable activity of phK in liver and peripheral blood cells, and XLG II with normal activity in peripheral blood cells and deficiency in the liver [8]. Although several female GSD IX patients with PHKA2 mutations have been reported in other populations, including Western and other Asian populations [18, 22–24], only male patients were identified among our Korean patient population. The PHKA2 mutation spectrum is known to be distributed across the entirety of PHKA2 exons, with the exception of exon 13. Except for amino acids 420, 423, and 432–434, the protein sequence of PHKA2 is strictly conserved among various mammals and zebrafish (Fig. 3). Exon 13, which corresponds to amino acids 416 to 442, is in a six-hairpin glycosidase domain (amino acids 9 – 453) that contains pathogenic mutations including several missense mutations [2]. Although several missense variant alleles have been identified in exon 13 (ExAC), hemizygosity and lack of clinical information make interpretation of their clinical significance difficult. Further studies are needed to clarify the clinical significance of mutations in exon 13.

No recurrent mutations were observed among Korean GSD IX patients, although a missense PHKA2 mutation of p.Pro1205Leu suggestive of a founder mutation has been reported among Dutch patients [22]. This mutation has also been reported in Japanese and Chinese patients, but has not been observed in the Korean population [22, 24, 25].

In this study, we identified one novel and five known PHKA2 mutations. The missense mutation of c.884G > A (p.Arg295His) has been reported in a patient with unusually severe clinical manifestation with marked ketosis and hyperlipidemia [8, 26] In our study, patient 2 carrying the same mutation showed a relatively mild clinical manifestation, with asymptomatic hepatomegaly and asymptomatic hypoglycemia and height and weight within the 25th to 50th percentiles, although he had experienced hyperlactic acidemia. This mutation has been reported previously in a patient with XLG I who had a pronounced PhK deficiency in both liver and erythrocytes (PhK activity was 10.0 % of the control mean in both liver and erythrocytes) and in a patient with XLG II who had normal erythrocyte PhK activity (83.0 % of the control mean), therefore the significance of this mutation for biochemical function in specific tissues and clinical severity remains speculative [8]. This mutation was predicted to affect protein function by in silico analyses (SIFT and PolyPhen-2) and was predicted to alter splicing through activation of an exonic cryptic donor site by both Human Splice Finder software and by a machine-learning technique that scores how strongly genetic variants affect RNA splicing [27]. The c.537 + 5G > A mutation has been reported in a male GSD IX patient of European ancestry who presented with hepatomegaly and growth delay that presented from 9 months of age [6]. This mutation was predicted to affect splicing through alteration of the wild-type donor site. Although in vitro analysis of the splicing effect of c.537 + 5G > A was not performed, in vivo results confirmed phosphorylase b kinase deficiency in the patient’s lymphocytes (32 U/g, normal range 100–240 U/g) and erythrocytes (0.7 U/g Hb, normal range 4–10 U/g Hb) [6]. Clinical manifestations of the patient carrying this mutation were reported to include growth delay, mild fasting hypoglycemia, post-prandial lactatemia, and elevated serum triglycerides, with age at onset of 9 months [6]. In our study, patient 1 carrying the same mutation had post-prandial lactatemia and elevated serum triglycerides in common, but no fasting hypoglycemia or growth delay. A small deletion mutation of c.3210_3212delGAG (p.Arg1072del) has been reported in a Finnish male GSD IX patient with hepatomegaly without short stature or hyperlactic acidemia [18] whose age at onset was 16 months. That patient was diagnosed with GSD IX by molecular diagnosis, but a PhK enzyme activity test was not performed [18]. In this study, the patient with the same mutation was of short stature and his liver glycogen concentrations increased over time (8.8 %/g wet liver weight at initial diagnosis and 22.5 %/g wet liver weight at 2.5 year follow-up; reference range 1–6 %/g wet liver weight). His age of onset was 4 years and he experienced hyperlactic academia, which was different from the Finnish male. A gross deletion mutation of exons 27–33 has been reported in a male GSD IX patient of European ancestry with PhK deficiency diagnosed by markedly decreased phosphorylase activities in his lymphocytes and erythrocytes, whose age at onset was 8 months [6]. Because PhK (defective PhK activities cause GSD IX) activates glycogen phosphorylase b (defective phosphorylase activities cause GSD VI) by phosphorylation, patients with GSD IX can have decreased phosphorylase activities [6]. Molecular studies and analysis of PhK are needed to make the correct diagnosis and avoid misdiagnosis of GSD VI [28]. The male patient in the previous literature was reported to have post-prandial lactatemia with elevated serum triglycerides [6]. In this study, patient 6 carrying the same large deletion had asymptomatic fasting hypoglycemia and post-prandial lactatemia but no elevated serum triglycerides. His age at first diagnostic workup was 4 years.

Of note, most mutations in Korean GSD IX patients were deletion or splicing mutations, except for one known missense mutation of c.884G > A (p.Arg295His) [8]. Fifty percent of Korean GSD IX patients had gross deletion mutations, and 83.3 % had splicing or deletion mutations that were different from those reported in other ethnic populations. These results suggest that laboratory tests for large deletions in PHKA2 should be included in the variety of methods that may be used such as quantitative PCR, long-range PCR, multiplex ligation-dependent probe amplification, and that chromosomal microarrays including this gene/chromosome segment should be used as the first approach for Korean patients suspected to have GSD IX [9]. In this study, the presence of deletions in male GSD IX patients was initially implied by the lack of amplification of exons by PCR and additional multiplex PCR was then was employed to better define the deletion. Although only male patients had PHKA2 mutations in this study, multiplex PCR would be necessary to detect deletions in females with suspected GSD IX [9]. Methods to detect deletions would be important for patients of all ethnicities [6, 9, 29].

Historically, diagnosis of GSD was based on enzymatic defects of each type of GSD or on liver histopathology compatible with GSD [6]. Recent molecular diagnostic approaches based on mutation analysis for disease-causing genes associated with each type of GSD provide the advantages of avoiding invasive liver biopsy and allowing differentiation between several types of GSD with similar clinical findings [6].

Because of its variable clinical manifestations with mild symptoms, underestimation of GSD IX is possible. For example, short stature was observed in 33.3 % of patients in this study, which was comparable to previous studies in European and Argentinian populations [18] but different from a Canadian study that reported no patients with short stature [3]. Furthermore, the effect of PHKA2 mutations on tissues other than liver should be clarified through future studies. A recent study reported that genetic diagnosis for X-linked mental retardation revealed several PHKA2 variants [30]. Although the natural history of GSD IX in Canadian patients has been reported, there are no reliable data on long-term outcomes for Korean GSD IX patients [3]. The study on 11 Canadian GSD IX patient with PHKA2 mutations with a follow-up period ranging from 1 to 16 years reported improved, normalized, or stable liver enzymes in all patients, and development of likely liver adenoma in one patient at the 5-year follow-up [3]. Detailed long-term natural history studies of Korean patients with GSD IX caused by PHKA2 mutation will be helpful to understand whether these patients are at increased risk of developing additional complications such as liver cirrhosis or hepatocellular carcinoma, or other conditions such as reproductive or mental disorders, later in life.

PhK deficiency is very complex and many genes play a key role in the GSD IX phenotype; the spectrum includes autosomal recessive forms of GSD IX (caused by mutations in PHKB, PHKG1, PHKG2, CALM1, CALM2, and CALM3) as well as the X-linked liver form (caused by mutations in PHKA2) and X-linked muscle form (caused by mutations in PHKA1). Only PHKA2 mutations were studied in this study. Future studies involving comprehensive mutational analyses of multiple causative genes in the Korean population are needed.

In this study we summarized the PHKA2 mutation spectrum in Korean GSD IX patients and found that the most common mutation type was gross deletion. The present study expands our knowledge of the mutational spectrum in Korean GSD IX patients, which differs from that in other ethnic populations. Considering the PHKA2 mutation spectrum in Korean patients with GSD IX, molecular diagnostic methods for deletions should be combined with direct sequence analysis to provide accurate molecular diagnosis of this disease.