Glycogen storage disease (GSD)
type 1a (von
Gierke disease) (OMIM 232200) is an inborn
error of metabolism caused by the deficiency
of glucose-6-phosphatase-a
in liver and kidney. Children present with short stature, failure to
thrive, hepatomegaly and symptoms of early morning hypoglycemia such as
drowsiness and seizures. Biochemical features include fasting
hypoglycemia, lactic acidosis, hypertriglyceridemia and hyperuricemia
[1]. Confirmation by biochemical method needs enzyme assay on liver
biopsy. Without differentiation between GSD types 1a, 1b and 3 may not
be always possible. The G6PC gene comprises of five exons and
sequencing the gene is known to reliably diagnose GSD type 1a with 100%
sensitivity and specificity [2]. We sequenced this gene in cases with
clinical, biochemical and histopathology features of GSD type 1a to
provide molecular diagnosis, genetic counselling and prenatal diagnosis
for the affected families.
Methods
Patients and their families were enrolled in this
study after obtaining an informed consent. The clinical history,
examination and investigation findings were recorded for the patients.
Essential clinical inclusion criteria included hepatomegaly with
glycogen accumulation demonstrated in liver histopathology, and
additional criteria were failure to thrive, early morning seizures,
fasting hypoglycemia, hyper-triglyceridemia, lactic acidosis and
hyperuricemia. Patients with elevated serum creatine phosphokinase
levels (more than 250 IU/l) were excluded since that suggested a
diagnosis of GSD type 3. Five mL of blood was collected from the
patients and their family members for the mutation analysis. DNA was
isolated from peripheral blood by standard salt extraction method. The
five coding exons and the exon-intron boundaries were amplified by
polymerase chain reaction (PCR) using previously published primers [3].
PCR steps were as previously described [4]. Sanger sequencing was
performed using ABI BigDye Terminator mix and automated sequencing was
performed on ABI3100 (Applied Biosystems, Foster city CA). This study
was approved by the institute’s Ethics Committee.
Results
Nine patients with glycogen storage disease were
evaluated during the study period, and the tenth case was reviewed
postmortem. Their clinical and biochemical features are summarized in
Table 1. Patient number 1 and 7 were diagnosed as GSD type 3
(elevated total serum creatine phosphokinase levels) and excluded from
the study. Bidirectional sequencing analysis for patient number 3 and 6
showed that they were homozygous for a novel substitution mutation
c.355C>G (p.H119D) (substitution of cytosine by guanine) (missense
mutation leading to substitution of histidine by aspartic acid). Parents
of patient number 6 were found to be heterozygous for the same mutation.
Samples from parents of patient number 3 were unavailable for analysis.
Both the affected patients were referred from Karnataka and were
unrelated. They belonged to Hindu families, both being products of
non-consanguineous marriages. Haplotype analysis to determine whether
this mutation could have originated from a common founder for both the
families could not be performed as parents of patient 3 were unavailable
for analysis. No mutations were found in the other five patients.
Parents of the tenth case were consanguineous. No mutations in G6PC
gene were detected in them.
Table 1 Clinical and Biochemical Features, and Result of Mutation Analysis in Patients
SN |
Age |
Sex |
LS |
TG |
UA |
Lac |
SG |
CPK |
LB |
Sequence change |
|
Yrs |
|
cm |
mg/dL |
mg/dL |
|
PT |
IU/L |
|
|
1 |
5 |
M |
12 |
220 |
6.6 |
N |
140 |
1400 |
G |
EX |
2 |
1.2 |
F |
8 |
360 |
7.8 |
NA |
50 |
140 |
G |
N |
3 |
1 |
F |
9 |
450 |
6.7 |
E |
50 |
200 |
G |
H119D.H119D |
4 |
4 |
M |
10 |
500 |
NA |
NA |
200 |
120 |
G |
N |
5 |
4 |
F |
14 |
240 |
5.2 |
NA |
50 |
140 |
G |
N |
6 |
1 |
F |
9 |
2262 |
7.1 |
E |
61 |
120 |
G |
H119D/H119D |
7 |
6 |
F |
8 |
880 |
5.8 |
N |
440 |
550 |
G |
EX |
8 |
3 |
F |
10 |
450 |
9 |
E |
387 |
140 |
G |
N |
9 |
0.5 |
M |
10 |
550 |
8.2 |
E |
220 |
125 |
G |
N |
10 |
0.5 |
M |
8 |
800 |
10.4 |
E |
150 |
110 |
G |
CN |
(SN: Patient ID, M: male, F: female, LS: Liver
span, TG: serum triglycerides, UA: serum uric acid, Lac: serum
lactate, NA: not available; E: elevated; N: Normal, SGPT: serum
glutamate pyruvate transaminase (IU/L), CPK: serum total
creatine phosphokinase, LB: liver biopsy; G: glycogen
accumulation, EX: excluded from sequencing analysis, CN: parents
detected not to be carriers of any G6PC mutation. All patients
had fasting hypoglycemia. None of these cases had neutropenia)
|
The mutation p.H119D was found to abolish the
restriction site of endonuclease EaeI similar to a previously
reported mutation viz p. H119L [5]. Approximately 500 ng of PCR
product of exon 3 from control individuals, affected patients and their
parents was digested by 0.5 U of enzyme (concentration 5 U/microliter).
Digested control PCR products showed two bands sized 100 base-pairs (bp)
and 200 bp, patients having mutation p.H119D showed only one band sized
300 bp and carrier parents showed three bands sized 300 bp, 200 bp and
100 bp on 2 % agarose gel electrophoresis.
Discussion
The above results show that GSD type 1a accounted for
20% (2/10 cases) of liver glycogenoses in our case series. The
prevalence of GSD type 1a amongst liver glycogenoses in India is very
similar to that observed in other studies [1,6]. Sequence analysis of
G6PC detects mutations in up to 100% of affected individuals in some
homogeneous populations but in mixed populations (eg, in the US)
detection rate is approximately 94% because both mutations could not be
detected in some individuals with clinically and enzymatically confirmed
GSD type [7]. This could be because deletion mutations of an exon(s) or
a whole gene are unlikely to be detected by sequence analysis. There
remains a possibility that the cases with no detectable G6PC
mutations were likely to be another type of GSD (type 3 or type 1b)
since those genes (AGL and G6PT, respectively) were not
analyzed [8].
To date 54 missense, 10 nonsense, 17
insertion/deletion and 3 splicing mutations have been identified in the
G6PC gene [2]. A previously known mutation p.H119L (histidine
replaced by leucine or C.356A>T) disrupts one of the four crucial
catalytic sites of the G6PC enzyme and leads to null enzyme
activity [2]. The mutation p.H119D can also be predicted to lead to null
enzyme activity. A previous study has demonstrated c.150_151delGT; a 2
base pair deletion in two unrelated Indian families in UK [9]. This
mutation was not demonstrated in our study.
It is expected that the mutations in Indian patients
may be different from those described in other populations. Knowing the
frequent mutations in a given population helps to provide a rapid cost
effective way to dignose these patients in a clinical setting. However,
till data on more patients is available, sequencing the whole gene is
the only option.
Acknowledgement: We thank Dr Ashwin Dalal and Dr
Padma Priya T from Centre for DNA Fingerprinting and Diagnostics,
Hyderabad for their assistance. We thank Dr Siddramappa J Patil from
Narayana Hrudayalaya Institute of Medical Sciences, Bangalore, Dr
Meenakshi Bhat from Centre for Human Genetics, Bangalore and Dr Mamta
Muranjan from Seth GS Medical College and KEM hospital, Mumbai for
referring patients. We also thank Indian Council of Medical Research for
the support for the DNA banking facility.
Contributors: All authors contributed to study
design, execution and drafting of the manuscript.
Funding: This project was supported by intramural
grant of Sanjay Gandhi Postgraduate Institute of Medical Sciences,
Lucknow. Competing interests: None stated.
What This Study Adds?
• GSD type 1a accounted for 2 out of our 10 cases of
clinically suspected liver glycogenoses.
|
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