Abstract
Genetic deficiency of the glycogen debranching enzyme causes glycogen storage disease type III, an autosomal recessive inherited disorder. The gene encoding this enzyme is designated as AGL gene. The disease is characterized by fasting hypoglycemia, hepatomegaly, growth retardation, progressive myopathy and cardiomyopathy. In the present study, we present clinical features and molecular characterization of two consanguineous Tunisian siblings suffering from Glycogen storage disease type III. The full coding exons of the AGL gene and their corresponding exon–intron boundaries were amplified for the patients and their parents. Gene sequencing identified a novel single point mutation at the conserved polypyrimidine tract of intron 21 in a homozygous state (IVS21-8A>G). This variant cosegregated with the disease and was absent in 102 control chromosomes. In silico analysis using online resources showed a decreased score of the acceptor splice site of intron 21. RT-PCR analysis of the AGL splicing pattern revealed a 7 bp sequence insertion between exon 21 and exon 22 due to the creation of a new 3′ splice site. The predicted mutant enzyme was truncated by the loss of 637 carboxyl-terminal amino acids as a result of premature termination. This novel mutation is the first mutation identified in the region of Bizerte and the tenth AGL mutation identified in Tunisia. Screening for this mutation can improve the genetic counseling and prenatal diagnosis of GSD III.
Similar content being viewed by others
References
Shen JJ, Chen YT (2002) Molecular characterization of glycogen storage disease type III. Curr Mol Med 2(2):167–175
Shen JJ, Bao Y, Liu HM, Lee P, Leonard JV, Chen YT (1996) Mutations in exon 3 of the glycogen debranching enzyme gene are associated with glycogen storage disease type III that is differentially expressed in liver and muscle. J Clin Invest 98(2):352–357
Van Hoof F, Hers HG (1967) The subgroups of type 3 glycogenosis. Eur J Biochem 2:265–270
Lucchiari S, Pagliarani S, Salani S, Filocamo M, Di Rocco M, Melis D, Rodolico C, Musumeci O, Toscano A, Bresolin N, Comi GP (2006) Hepatic and neuromuscular forms of glycogenosis type III: nine mutations in AGL. Hum Mutat 27(6):600–601. doi:10.1002/humu.9426
Okubo M, Horinishi A, Nakamura N, Aoyama Y, Hashimoto M, Endo Y, Murase T (1998) A novel point mutation in an acceptor splice site of intron 32 (IVS32 A-12–>G) but no exon 3 mutations in the glycogen debranching enzyme gene in a homozygous patient with glycogen storage disease type IIIb. Hum Genet 102(1):1–5
Yang BZ, Ding JH, Enghild JJ, Bao Y, Chen YT (1992) Molecular cloning and nucleotide sequence of cDNA encoding human muscle glycogen debranching enzyme. J Biol Chem 267(13):9294–9299
Parvari R, Moses S, Shen J, Hershkovitz E, Lerner A, Chen YT (1997) A single-base deletion in the 3′-coding region of glycogen-debranching enzyme is prevalent in glycogen storage disease type IIIA in a population of North African Jewish patients. Eur J Hum Genet 5(5):266–270
Santer R, Kinner M, Steuerwald U, Kjaergaard S, Skovby F, Simonsen H, Shaiu WL, Chen YT, Schneppenheim R, Schaub J (2001) Molecular genetic basis and prevalence of glycogen storage disease type IIIA in the Faroe Islands. Eur J Hum Genet 9(5):388–391. doi:10.1038/sj.ejhg.5200632
Aoyama Y, Ozer I, Demirkol M, Ebara T, Murase T, Podskarbi T, Shin YS, Gokcay G, Okubo M (2009) Molecular features of 23 patients with glycogen storage disease type III in Turkey: a novel mutation p.R1147G associated with isolated glucosidase deficiency, along with 9 AGL mutations. J Hum Genet 54(11):681–686. doi:10.1038/jhg.2009.100
Okubo M, Aoyama Y, Murase T (1996) A novel donor splice site mutation in the glycogen debranching enzyme gene is associated with glycogen storage disease type III (vol 224, pg 493, 1996). Biochem Biophys Res Commun 225(2):695
Okubo M, Horinishi A, Suzuki Y, Murase T, Hayasaka K (2000) Compound heterozygous patient with glycogen storage disease type III: identification of two novel AGL mutations, a donor splice site mutation of Chinese origin and a 1-bp deletion of Japanese origin. Am J Med Genet 93(3):211–214
Okubo M, Horinishi A, Takeuchi M, Suzuki Y, Sakura N, Hasegawa Y, Igarashi T, Goto K, Tahara H, Uchimoto S, Omichi K, Kanno H, Hayasaka K, Murase T (2000) Heterogeneous mutations in the glycogen-debranching enzyme gene are responsible for glycogen storage disease type IIIa in Japan. Hum Genet 106(1):108–115
Shen JJ, Bao Y, Chen YT (1997) A nonsense mutation due to a single base insertion in the 3′-coding region of glycogen debranching enzyme gene associated with a severe phenotype in a patient with glycogen storage disease type IIIa. Hum Mutat 9(1):37–40
Endo Y, Horinishi A, Vorgerd M, Aoyama Y, Ebara T, Murase T, Odawara M, Podskarbi T, Shin YS, Okubo M (2006) 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 51(11):958–963. doi:10.1007/s10038-006-0045-x
Lucchiari S, Fogh I, Prelle A, Parini R, Bresolin N, Melis D, Fiori L, Scarlato G, Comi GP (2002) Clinical and genetic variability of glycogen storage disease type IIIa: seven novel AGL gene mutations in the Mediterranean area. Am J Med Genet 109(3):183–190. doi:10.1002/ajmg.10347
Horinishi A, Okubo M, Tang NL, Hui J, To KF, Mabuchi T, Okada T, Mabuchi H, Murase T (2002) Mutational and haplotype analysis of AGL in patients with glycogen storage disease type III. J Hum Genet 47(2):55–59. doi:10.1007/s100380200000
Maire I, Baussan C, Moatti N, Mathieu M, Lemonnier A (1991) Biochemical diagnosis of hepatic glycogen storage diseases: 20 years French experience. Clin Biochem 24(2):169–178
Desmet FO, Hamroun D, Lalande M, Collod-Beroud G, Claustres M, Beroud C (2009) Human Splicing Finder: an online bioinformatics tool to predict splicing signals. Nucleic Acids Res 37(9):e67. doi:10.1093/nar/gkp215
Reese MG, Eeckman FH, Kulp D, Haussler D (1997) Improved splice site detection in Genie. J Comput Biol 4(3):311–323
Blanco E, Parra G, Guigó R (2007) Using geneid to identify genes. Curr Protoc Bioinformatics 4(4.3):1–28
Yeo G, Burge CB (2004) Maximum entropy modeling of short sequence motifs with applications to RNA splicing signals. J Comput Biol 11(2–3):377–394
Momoi T, Sano H, Yamanaka C, Sasaki H, Mikawa H (1992) Glycogen storage disease type III with muscle involvement: reappraisal of phenotypic variability and prognosis. Am J Med Genet 42(5):696–699. doi:10.1002/ajmg.1320420514
Lee P, Burch M, Leonard JV (1995) Plasma creatine kinase and cardiomyopathy in glycogen storage disease type III. J Inherit Metab Dis 18(6):751–752
Coleman RA, Winter HS, Wolf B, Gilchrist JM, Chen YT (1992) Glycogen storage disease type III (glycogen debranching enzyme deficiency): correlation of biochemical defects with myopathy and cardiomyopathy. Ann Intern Med 116(11):896–900
Baralle D, Baralle M (2005) Splicing in action: assessing disease causing sequence changes. J Med Genet 42(10):737–748. doi:10.1136/jmg.2004.029538
Krawczak M, Reiss J, Cooper DN (1992) The mutational spectrum of single base-pair substitutions in mRNA splice junctions of human genes: causes and consequences. Hum Genet 90(1–2):41–54
Crispino JD, Mermoud JE, Lamond AI, Sharp PA (1996) Cis-acting elements distinct from the 5′ splice site promote U1-independent pre-mRNA splicing. RNA 2(7):664–673
Hadjigeorgiou GM, Comi GP, Bordoni A, Shen J, Chen YT, Salani S, Toscano A, Fortunato F, Lucchiari S, Bresolin N, Rodolico C, Piscaglia MG, Franceschina L, Papadimitriou A, Scarlato G (1999) Novel donor splice site mutations of AGL gene in glycogen storage disease type IIIa. J Inherit Metab Dis 22(6):762–763
Lucchiari S, Donati MA, Melis D, Filocamo M, Parini R, Bresolin N, Comi GP (2003) Mutational analysis of the AGL gene: five novel mutations in GSD III patients. Hum Mutat 22(4):337. doi:10.1002/humu.9177
Mili A, Ben Charfeddine I, Amara A, Mamai O, Adala L, Ben Lazreg T, Bouguila J, Saad A, Limem K, Gribaa M (2012) A c.3216_3217delGA mutation in AGL gene in Tunisian patients with a glycogen storage disease type III: evidence of a founder effect. Clin Genet 82(6):534–539. doi:10.1111/j.1399-0004.2011.01806.x
Mili A, Ben Charfeddine I, Mamai O, Abdelhak S, Adala L, Amara A, Pagliarani S, Lucchiarri S, Ayadi A, Tebib N, Harbi A, Bouguila J, H’Mida D, Saad A, Limem K, Comi GP, Gribaa M (2012) Molecular and biochemical characterization of Tunisian patients with glycogen storage disease type III. J Hum Genet 57(3):170–175. doi:10.1038/jhg.2011.122
Cherif W, Ben Rhouma F, Messai H, Mili A, Gribaa M, Kefi R, Ayadi A, Boughamoura L, Chemli J, Saad A, Kaabachi N, Sfar MT, Ben Dridi MF, Tebib N, Abdelhak S (2012) High frequency of W1327X mutation in glycogen storage disease type III patients from central Tunisia. Ann Biol Clin (Paris) 70(6):648–650. doi:10.1684/abc.2012.0766
Acknowledgments
The authors are extremely grateful to the patients and their family whose participation made this work possible. The authors confirm independence from the sponsors; the content of the article has not been influenced by the sponsors. The study was supported by the Tunisian Ministry of Public Health and the Ministry of Higher education and Scientific Research.
Conflict of interest
The authors declare that they have no competing interests.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Ben Rhouma, F., Azzouz, H., Petit, F.M. et al. Molecular and biochemical characterization of a novel intronic single point mutation in a Tunisian family with glycogen storage disease type III. Mol Biol Rep 40, 4197–4202 (2013). https://doi.org/10.1007/s11033-013-2500-z
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s11033-013-2500-z