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.

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

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.

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.

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