Novel AGL mutation in a Turkish patient with glycogen storage
disease type IIIaped_2943145..••
Yoshiko Aoyama,1Yoriko Endo,1Tetsu Ebara,1Toshio Murase,1Yoon S. Shin,2Teodor Podskarbi,2Isil Ozer,3
Mübeccel Demirkol,3Gülden Gökçay3and Minoru Okubo1,4
1Okinaka Memorial Institute for Medical Research,4Department of Endocrinology and Metabolism, Toranomon Hospital,
Tokyo, Japan,2Molecular Genetics and Metabolism Laboratory, Munich, Germany and3Department of Pediatric Nutrition
and Metabolism, Istanbul Medical Faculty, Istanbul University, Istanbul, Turkey
AGL, deletion, glycogen debranching enzyme, glycogen storage disease type IIIa, Turkey.
Glycogen storage diseases (GSD) are heterogeneous groups of
inborn errors of glycogen metabolism and are divided into
several types based on deficient enzymes.1,2Glycogen is metabo-
lized by glycogen phosphorylase and glycogen debranching
enzyme (GDE). GDE is a single protein with two independent
catalytic activities: oligo-1, 4-1, 4 glucantransferase (EC
22.214.171.124) and amylo-1, 6-glucosidase (AGL) (EC 126.96.36.199). The
gene encoding GDE is designated as the AGL gene. Deficiency of
GDE is GSD type III (GSD III; OMIM 232400), an autosomal
recessive inherited disorder. The disease is characterized by
fasting hypoglycemia, growth retardation, and hepatomegaly.
Most GSD III patients are GDE deficient in both liver and muscle
(type IIIa) and present muscle weakness in adulthood, whereas
15% of patients have GDE absent in liver but present in muscle
tissue (type IIIb) and do not show any muscular symptoms.These
clinical manifestations are almost indistinguishable from those in
GSD type Ia (OMIM 232200), glucose-6-phosphatase deficiency.
Long-term complications, however, are different between the two
GSD types. GSD IIIa involves cardiomyopathy,3liver cirrhosis,
and hepatocellular carcinoma,4while gout, hepatic adenomas,
osteoporosis, and renal disease are present in patients with GSD
Ia. Clear differential diagnosis of the two hepatic GSD requires
both an enzymatic and a molecular assessment.
Here, we present a patient with GSD IIIa who was referred to
Istanbul University Hospital as having GSD Ia, and describe a
novel AGL mutation in this patient of Turkish ancestry.
The present patient was an 11-year-old Turkish boy. He was born
at full term and hepatomegaly was noticed at the age of 7 months.
A liver biopsy, performed in a hospital in Turkey at the age of 1
year, showed elevated glycogen content, enlarged hepatocytes
with glycogen, and mild portal fibrosis. He was then diagnosed as
having ‘GSD Ia’and had been on dietary therapy with corn starch
to prevent hypoglycemia.
The patient was referred to Istanbul University when he was
31/2years old. At the first evaluation he had short stature (height
86 cm, <3rd percentile), a doll-like face, hepatomegaly, and sple-
nomegaly. Laboratory data showed elevated liver enzymes
(aspartate aminotransferase, 718 IU/L; alanine aminotransferase,
333 IU/L), hypercholesterolemia (total cholesterol, 278 mg/dL),
and hypertriglyceridemia (triglyceride, 665 mg/dL). The patient
had chronic hypochromic normocytic anemia (hemoglobin,
8.8 g/dL), which gradually resolved. His plasma lactate level
fluctuated between 63.1 and 7.2 mg/dL(normal range, <19.8 mg/
dL). In contrast, fasting plasma glucose, uric acid, and creatine
phosphokinase (CPK) levels were within normal range (106 mg/
dL, 3.6 mg/dL, and 49 IU/L, respectively). An echocardiogram
was normal at that time.
Diagnosis of ‘GSD Ia’ in the patient did not fit the clinical
data: he had higher liver enzymes and glucose levels and lower
levels of uric acid, lactate, and lipid concentrations than the
average in Turkish patients with GSD Ia.5Moreover, serum bio-
tinidase activity was normal (5.2 nmol/min per mL; normal
range, 3.1–10.2 nmol/min per mL).
His clinical manifestations prompted us to speculate that he
may have GSD III. The patient’s GDE activity was undetectable
(normal range: 0.6–3.5 nmol/min per g Hb) in peripheral blood
cells. He has been on a special diet involving carbohydrate (50–
55% energy requirements) and protein enrichment (20% energy
requirement) and required continuous drip feeding at night after
the referral to hospital. He has had a gastrostomy since the age of
6 years. Improvement of liver enzymes and lipid levels was
evident.As he grew older, CPK increased to 955 IU/L. The latest
echocardiogram showed that he had left ventricular hypertrophy.
Ultrasonography showed that the patient had hepatomegaly and
normal echogenicity of the liver. Family history indicated that his
parents were first cousins. Parents and his sibling had no indica-
tions of GSD.
Molecular analysis was approved by the hospital ethics com-
mittee and informed consent was obtained from his parents.
Sequencing analysis of the patients’ AGL was performed as
described previously.6A novel deletion of C at nucleotide 2474
(c.2474delC) was identified in exon 20 (Fig. 1a,b).There were no
other mutations in the patient. The mutation identified in the
patient was verified using restriction fragment length polymor-
phism (Fig. 1c). The patient was a homozygote for c.2474delC
mutation, and the parents were heterozygotes. Of 50 Turkish
controls examined, none had the mutation. AGL haplotype was
determined as reported previously.7The haplotype associated
Correspondence: Minoru Okubo, MD, PhD, Okinaka Memorial Insti-
tute for Medical Research and Toranomon Hospital, 2-2-2 Toranomon,
Minato-ku, Tokyo 105-8470, Japan. Email: email@example.com
Received 3 January 2008; revised 17 April 2008; accepted 27 May
Novel mutation in Turkish GSD IIIa145
© 2010 Japan Pediatric Society
with c.2474delC was different from those for two other mutations
in Turkish patients (Table 1): in four single nucleotide polymor-
phisms (-10 in exon 3/IVS3 + 85/IVS6-73/L298L), the present
patient had (g/t/g/t), while the two other Turkish patients had
The present patient was found to be deficient in GDE activity and
homozygous for a novel 1 bp deletion in AGL. This mutation is
predicted to cause premature termination at codon 834 due to
frame shift, resulting in an inactive enzyme owing to the loss of
an important domain for glycogen binding at the carboxyl termi-
nal. To date, >50 different mutations have been reported in AGL,
and the spectrum of AGL mutations in GSD IIIa varies among
ethnic groups.8Specific mutations are prevalent in subjects of
North African Jewish decent and in an isolated area such as the
Faroe Islands. In contrast, genetic heterogeneity has been shown
in other ethnic groups, such as Japanese subjects.6As for AGL
mutations in Turkish patients, two others have already been
reported7and in the present study we report a third. A further
genetic survey is under way at Okinaka Memorial Institute for
Medical Research to explore AGL mutations in Turkish GSD III
Clinical indications for differential diagnosis of the two
hepatic GSD are as follows: in GSD IIIa, (i) higher concentra-
tions of liver enzymes and glucose; (ii) no elevated biotinidase
activity; and (iii) splenomegaly. CPK may be a useful marker for
diagnosis of GSD III when elevated; the patient’s level was,
however, normal at 31/2 years of age. As the patient grew older,
(iv) elevated CPK levels and cardiomyopathy become prominent.
Biotinidase activity has been shown to be higher in most GSD Ia
patients.9Recently, Paesold-Burda et al. reported that sensitivity
of increased biotinidase activity was 100% for GSD Ia patients
and 62% for GSD III patients.10Splenomegaly was not observed
20. (a)The patient was homozygous for a novel deletion c.2474delC.
(b) Normal sequence from a control (arrow, C at nucleotide 2474).
(c) Electrophoresis of polymerase chain reaction (PCR) products
after digestion with Bsl I, which verified that the patient was a
homozygote for c.2474delC, and that parents and a sibling were
heterozygotes. M, DNA marker; 1, patient; 2, mother; 3, father; 4,
sibling; 5, control. Sequences surrounding the point mutation were
amplified using a sense primer (5′-tca tct tac tac tgt gtt tag c-3′) and
an antisense primer (5′-agt aaa gtc aac agt ttg agc-3′). Restriction
endonuclease Bsl I was added to digest PCR products. In the normal
sequence the 228 bp product was cleaved into 125 bp and 103 bp
fragments, but in the presence of the C deletion it was uncleaved and
227 bp long. Restriction digests were analyzed using a 5% polyacry-
(a,b) Sequence electropherogram of the sense strand in exon
AGL mutation and SNP in Turkish patients with GSD IIIa
IVS7 + 5G > A
IVS7 + 5G > A
IVS21 + 5insA
IVS21 + 5insA
-10 in exon3
IVS3 + 85
-2 in Ex4′
IVS12 + 74
IVS16 + 8
IVS21 + 124
IVS22 + 11
IVS29 + 45
IVS29 + 53
†Reported by Endo et al. (2006).7
GSD, glycogen storage disease; n.d., not determined; SNP, single
146Y Aoyama et al.
© 2010 Japan Pediatric Society
in the Turkish GSD Ia patients, but 70% of GSD III patients had
splenomegaly.5Accordingly, clinical evaluation suggested that
typing of GSD should be reconsidered in the present patient,
which was confirmed on enzymatic and molecular analysis.
In conclusion, the present case underscores clinical indica-
tions for differential diagnosis between GSD IIIa and GSD Ia and
the importance of enzymatic and molecular analysis.
This study was supported in part by Japan Society for the Pro-
motion of Science and the Nakatomi foundation.
1 Chen YT. Glycogen storage diseases. In: Scriver CR, Beaudet AL,
Sly WS, Valle D (eds). The Metabolic and Molecular Bases of
Inherited Disease, 8th edn, Vol. I. McGraw-Hill, NewYork, 2001;
2 Shin YS. Glycogen storage disease: Clinical, biochemical, and
molecular heterogeneity. Semin. Pediatr. Neurol. 2006; 13: 115–
3 Ogimoto A, Okubo M, Okayama H et al. Japanese patient with
cardiomyopathy caused by a novel mutation R285X in the AGL
gene. Circ. J. 2007; 71: 1653–6.
4 Demo E, Frush D, Gottfried M et al. Glycogen storage disease type
III-hepatocellular carcinoma a long-term complication? J. Hepatol.
2007; 46: 492–8.
5 Demirkol M, Hüner G, Baykal T, S ¸arbat G, Özenog ˘lu A, Shin YS.
Hepatic glycogen storage disease in Istanbul. In: Demirkol M, Shin
YS (eds). Diagnosis and Treatment of Inborn Errors of Metabo-
lism: Contributions to an Equal Opportunity for Children in Asia
and Europe. Ufuk Press, Istanbul, 1996; 136–43.
6 Okubo M, Horinishi A, Takeuchi M et al. Heterogeneous muta-
tions in the glycogen-debranching enzyme gene are responsible for
glycogen storage disease type IIIa in Japan. Hum. Genet. 2000;
7 Endo Y, Horinishi A, Vorgerd M et al. Molecular analysis of the
AGL gene: Heterogeneity of mutations in patients with glycogen
storage disease type III from Germany, Canada, Afghanistan, Iran,
and Turkey. J. Hum. Genet. 2006; 51: 958–63.
8 Shen JJ, Chen YT. Molecular characterization of glycogen storage
disease type III. Curr. Mol. Med. 2002; 2: 167–75.
9 Burlina AB, Dermikol M, Mantau A et al. Increased plasma bio-
tinidase activity in patients with glycogen storage disease type Ia:
Effect of biotin supplementation. J. Inherit. Metab. Dis. 1996; 19:
10 Paesold-Burda P, Baumgartner MR, Santer R, Bosshard NU,
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a1-Antichymotrypsin deficiency associated with liver cirrhosisped_2950147..••
Luis Ortega,1Florencio Balboa2and Lucía González1
Departments of1Pathology and2Pediatrics, San Carlos Clinical Hospital, Madrid, Spain
a1-Antichymotrypsin deficiency, cirrhosis, serpin.
a1-Antichymotrypsin (ACT) and a1-antitrypsin (AAT) are serine
proteinase inhibitors of the serpin family.ACT is highly homolo-
gous to AAT, both in aminoacid sequence and in gene arrange-
ment, although their deficiency occurs independently.1
a1-Antichymotrypsin deficiency is found in approximately
0.6% of the Swedish population. The abnormal gene is inherited
in an autosomal dominant way and no homozygotes have yet
a1-Antitrypsin deficiency is clearly associated with lung and
liver disease, and ACT deficiency can cause the same pathologi-
cal changes.3The increase in the prevalence of liver disease
(chronic hepatitis and cryptogenic cirrhosis) inACTdeficiency is
well known in adulthood, but to our knowledge no case in chil-
dren has been reported to date.
A 3-year 8-month-old boy was admitted to San Carlos Clinical
Hospital to assess liver transplantation because of cirrhosis. The
patient was a native of Venezuela and had been diagnosed with
liver cirrhosis secondary to AAT deficiency. The boy had born
after a full-term pregnancy to a 31-year-old woman (gravida 3,
para 3) in an euthocic delivery. Family history was unremarkable,
two brothers were healthy, a maternal aunt suffered from
a-thalassemia and the paternal grandfather from uncontrolled
The boy had been suffering from recurrent episodes of upper
airway infections since the age of 3 months. From the age of 20
months he developed jaundice, growth retardation and abdominal
At age 3 years 8 months the patient was pale and weighed
14.4 kg (25th–50th percentile). Abdominal palpation indicated a
non-tender, hard liver descending 7–8 cm below the right costal
margin. The spleen was soft, descending 4–5 cm below the costal
margin. There was no ascites.
Hematological and coagulation values are listed in Table 1,
blood chemistry is detailed in Table 2. Copper in 24 h urine was
16.5 mg/dL(normal: 3–35 mg/dL). Serologic tests were negative
Correspondence: Luis Ortega, PhD, Servicio de Anatomía Patológica,
Hospital Clínico San Carlos, C/ Martín Lagos sn. 28040, Madrid,
Spain. Email: firstname.lastname@example.org
Received 19 January 2006; revised 17April 2008; accepted 17 June
Novel mutation in Turkish GSD IIIa147
© 2010 Japan Pediatric Society
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