==== Front Ann Saudi MedAnn Saudi MedAnnals of Saudi Medicine0256-49470975-4466King Faisal Specialist Hospital and Research Centre 2582769510.5144/0256-4947.2014.390asm-5-390Original ArticleA founder splice site mutation underlies glycogen storage disease type 3 in consanguineous Saudi families Basit Sulman aMalibari Omhani bAl Balwi Alia Mahmood aAbdusamad Firoz aIsmail Feras Abu c a Center for Genetics and Inherited Diseases, Taibah University Almadinah Almunawarah, Saudi Arabia b Department of Metabolic Diseases, King Abdullah Medical City-Madinah Maternity and Children Hospital, Almadinah Almunawarah, Saudi Arabia c Al Nakheel Hospital, Nakheel, Almadinah Almunawarah, Saudi ArabiaCorrespondence: Sulman Basit PhD, Center for Genetics and Inherited Diseases, Taibah University Al Madinah Al Munawarah, Saudi Arabia, T: +966-535370209, sbasit.phd@gmail.comSep-Oct 2014 34 5 390 395 Copyright © 2014, Annals of Saudi Medicine2014This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.BACKGROUND AND OBJECTIVES Glycogen storage disease type 3 (GSD III) is an autosomal recessive disorder caused by genetic mutations in the gene AGL. AGL encodes amylo-α-1, 6-glucosidase, 4-α-glucanotransferase, a glycogen debranching enzyme. GSD III is characterized by fasting hypoglycemia, hepatomegaly, growth retardation, progressive myopathy, and cardiomyopathy due to storage of abnormally structured glycogen in both skeletal and cardiac muscles and/or liver. The aim of this study is to detect mutations underlying GSD III in Saudi patients. DESIGN AND SETTINGS A cross-sectional clinical genetic study of 5 Saudi consanguineous families examined at the metabolic clinic of the Madinah Maternity and Children Hospital. PATIENTS AND METHODS We present a biochemical and molecular analysis of 5 consanguineous Saudi families with GSD III. DNA was isolated from the peripheral blood of 31 individuals, including 12 patients, and the AGL gene was sequenced bidirectionally. DNA sequences were compared with the AGL reference sequence from the ensemble genome browser. RESULTS Genotyping and sequence analysis identified a homozygous intronic splice acceptor site mutation (IVS32-12A>G) in 4 families perfectly segregating with the phenotype. Complementary (c)DNA sequence analysis of the AGL gene revealed an 11-bp sequence insertion between exon 32 and exon 33 due to the creation of a new 3′ splice site. The predicted mutant enzyme was truncated by 112 carboxyl-terminal amino acids as a result of premature termination. CONCLUSION Haplotype analysis revealed that the mutation arises as a result of founder effect, not an independent event. This is the first report of a genetic mutation in the AGL gene from Saudi Arabia. Screening for this mutation can improve genetic counseling and prenatal diagnosis of GSD III in Saudi Arabia. ==== Body Glycogen storage diseases (GSDs) comprise at least 16 different inherited conditions caused by genetic mutations in the genes encoding enzymes that regulate the synthesis or degradation of glycogen. Glycogen storage disease type 3 (GSD III) is an autosomal recessive metabolic disorder characterized by fasting hypoglycemia, hepatomegaly with increased transaminases, growth retardation, progressive myopathy, and cardiomyopathy with elevated creatine phosphokinase.1 GSD III has 2 major subtypes: GSD IIIa and GSD IIIb. GSD IIIa affects both the liver and muscles and accounts for 80% of GSD III cases, while GSD IIIb affects only the liver; the patient does not show any muscular manifestation. GSD IIIb accounts for approximately 15% of GSD III cases.2 During childhood, it is difficult to differentiate between GSD IIIa and GSD IIIb, as the muscular manifestation starts together with liver disorders, or muscular symptoms arise in adulthood because of the progressive accumulation of glycogen in the muscles.3,4 There is no specific treatment for GSD, and GSD III patients require lifelong follow-up.5 Diet therapy improves symptoms (especially hypoglycemia), reduces the liver size, and assists with the resumption of overall growth and development.6 GSD III results from genetic mutations in the AGL gene. The AGL gene is located on chromosome 1p21.2, spanning 85 kb, and consists of 35 exons (UCSC genome browser). The AGL gene encodes amylo-α-1, 6-glucosidase, 4-α-glucanotransferase, a glycogen debranching enzyme (GDE) of 1515 amino acids in length. The GDE has 2 catalytic domains performing 2 independent catalytic activities: oligo1,4-glucotransferase (EC 2.4.1.25) and amylo-1,6-glucosidase (EC3.2.1.33). Both catalytic sites are required for a complete action of the debranching enzyme.7 GDE is expressed mainly in the liver and muscles of the heart and skeleton. GDE deficiency causes an excessive accumulation of abnormal glycogen and results in the onset of GSD III. Homozygous knockout mice for GDE recapitulate the human GSD III phenotype, and it has been shown that repeated fasting is detrimental to these mice.8 Molecular studies have confirmed that GSD III is genetically highly heterogeneous with a large spectrum of mutations in the same population.4,9,10 Recurrent mutations have also been reported in populations with high consanguinity.11 In Saudi Arabia, the rate of consanguinity is 56%, which contributes greatly to the high frequency of autosomal recessive disorders in the country,12 but the prevalence of AGL mutations in GSD III is still unknown. We located 5 Saudi families with GSD III and performed a molecular study by screening the AGL gene in all affected and unaffected members of the families. We identified a homozygous splice site mutation in the AGL gene in 4 families. Complementary (c)DNA sequencing revealed an 11-bps insertion in the patients’ messenger (m)RNA. Haplotype analysis identified that the splice site mutation is due to the founder effect and not an independent event. PATIENTS AND METHODS Families In the present study, 5 consanguineous Saudi families (A, B, C, D, and E) originating from Madinah in western Saudi Arabia were investigated. Thirty-one individuals, including 12 affected, volunteered to participate in the study (Figure 1). The age of onset of the disease was 9 months to 3 years. Prior to the start of the study, approval was obtained from the ethical committee of Madinah Maternity and Children Hospital. Informed consent was obtained from all subjects participating in the study. All affected individuals showed typical features of autosomal recessive GSD IIIb with no other abnormalities, and their growth conditions were normal. Genomic DNA and Total RNA Isolation Blood samples for DNA and RNA isolation were collected in ethylenediaminetetraacetic acid (EDTA)-containing Vacutainer sets (BD, Franklin, NJ, USA) and Tempus Blood RNA tubes, respectively (Applied Biosystems, Foster City, CA, USA) from both affected and unaffected individuals of the 5 families. Genomic DNA was extracted using the QIAamp DNA Mini Kit (Qiagen Inc., Valencia, CA, USA). Total RNA was extracted using Tempus Spin RNA Isolation Kit (Applied Biosystems). To quantify DNA and RNA, a MaestroNano spectrophotometer (Green BioResearch, Los Angeles, CA, USA) was used, measuring optical density at 260 nm and diluted to 40–50 ng/μL for amplification by polymerase chain reaction (PCR). PCR products were resolved on 2% agarose gel stained with ethidium bromide. Screening the AGL gene It has been reported that mutations in the AGL gene result in GSD III. Therefore, all coding exons and adjacent sequences of intron–exon borders were amplified by PCR using gene-specific primers. PCR primers were designed using the Primer3 program (http://frodo.wi.mit.edu/primer3) and checked for specificity using a basic local alignment search tool (http://genome.ucsc.edu/cgi-bin/hgBlat). The amplification conditions used were 95°C for 1 minute, followed by 30 cycles of 95°C for 35 seconds, 60°C for 35 seconds, and 70°C for 3.5 minutes, followed by a single incubation at 70°C for 10 minutes. The PCR products were purified using the QIAquick PCR Purification Kit (Qiagen Inc.) and sequenced bidirectionally following dideoxy chain termination chemistry using BigDye Terminator v3.1 Cycle Sequencing Kit on AB 3500 genetic analyzer (Applied Biosystems) according to the manufacturer’s instructions. cDNA sequencing cDNA was synthesized from total RNA using the ProtoScript First Strand cDNA Synthesis Kit (New England Biolabs, Ipswich, MA, USA). Reverse transcription (RT) was carried out with 200 ng of total RNA. Hybridization of the oligo(dT) was realized by incubating the following mix for 5 minutes at 70°C: 3 μL of RNA; 2 μL of polyT oligo primers (dT) (10 mM, New England Biolabs); and 3 μL of H2O (RNase free), followed by ice quenching. RT was then carried out for 60 minutes at 42°C after the addition of 2 μL of M-MuLV enzyme mix and 10 μL of M-MuLV reaction mix (New England Biolabs). For the subsequent PCR, 5 μL of the obtained cDNA mix was used. Primers were designed to amplify exons 30–33 from cDNA to characterize the consequences of the splice acceptor site mutation. The reversed transcribed RNA was amplified with these primers (40 cycles) at a hybridization temperature of 57°C. Samples were then sequenced on an ABI 3500 DNA sequencer (Applied Biosystems). Genotyping with microsatellite markers Four microsatellite markers (D1S2671, D1S1658, D1S2767, and D1S2896) flanking the AGL gene on chromosome 1 were used to trace the haplotype surrounding the AGL gene in all 5 families. PCR amplification conditions for microsatellite markers were the same as described earlier by Basit et al.13 The allele size for the respective microsatellite markers were determined using 05 bp, 10 bp, and 20 bp DNA ladders (MBI, Fermentas, York, UK). RESULTS Patients The index case is a 9-year-old female (Figure 1A, II: 3). She was discovered and diagnosed as a GSD III patient at the age of 1 year by screening for hepatomegaly. Laboratory findings revealed fasting hypoglycemia and elevated transaminase (creatine phosphokinase 436 IU/L, alkaline phosphatase 409 IU/L). The lipid level was in the normal range (cholesterol: 3.9 mmol/L, triglycerides: 1.79 mmol/L). Hepatomegaly was confirmed with abdominal ultrasound. Cardiomyopathy was ruled out by echocardiography at the age of 8 months. Hypoglycemia was controlled using corn starch. Affected individuals in all 5 families exhibited features of GSD IIIb. The ages of the affected members ranged from 2–50 years at the time of the study. Table 1 shows the clinical and biochemical findings of the affected individuals of the families. Mostly similar clinical features were observed in the affected members of all 5 families (Table 1). Affected individuals had no clinical and biochemical evidence of muscle involvement. Sequencing the AGL gene in affected and normal individuals The AGL gene located on chromosome 1p21.2 is the only gene known to be responsible for generating the GSD III phenotype when mutated. To search for the underlying mutation, all 35 exons of the AGL gene and their splice junctions were PCR amplified from the genomic DNA of affected and unaffected individuals of the family, using primers designed from intronic sequences of the gene. PCR products were purified using the QIAquick PCR Purification Kit (Qiagen Inc.) and were sequenced in an ABI 3500 automated DNA sequencer using the Big Dye Terminator Cycle Sequencing Kit (PE Applied Biosystems, Foster city, CA, USA). Sequence analysis of the PCR-amplified products revealed a homozygous splice acceptor site mutation (VS32-12A>G) in all affected individuals of 4 families (A, C, D, and E). This mutation is present in a heterozygous state in parents (Figure 2). In family B, sequence analysis failed to detect any pathogenic variant that could be responsible for the GSD III phenotype. Sequencing of cDNA of the AGL gene in affected and normal individuals Amplification of exons 30 to 33 from the cDNA of affected individuals of 4 families (A, C, D, and E) was carried out using primers designed in the coding part of the AGL gene. Sequencing followed by alignment with the reference sequence revealed an insertion of 11 bps in the mRNA sequence of the AGL gene in affected individuals (Figure 2). Genotyping and haplotype construction Genotyping with 4 microsatellite markers flanking the AGL gene was carried out. Genotyping data and haplotype analysis revealed a similar homozygous pattern of the allele and an identical haplotype surrounding the AGL mutation in the affected individuals of 4 families (A, C, D, and E). Heterozygous haplotypes were obtained for the affected individuals of family B. DISCUSSION The diagnosis of GSD III requires the combination of clinical, biochemical, and molecular investigations. The broad spectrum of mutations in the AGL gene underlying GSD III and the large size of the AGL gene makes genetic diagnosis difficult. The genetic study of a specific population would help to identify inherited mutations due to the founder effect, generation after generation. We started to screen for the AGL gene in GSD III patients in Saudi Arabia to identify specific genetic variants common to this population. In this study, we investigated 5 families with 16 GSD III patients. Most of them have manifested fasting hypoglycemia and/or hepatomegaly. Cardiomyopathy was excluded in most of the patients with echocardiography. Clinical and biochemical data of the patients presented here rule out muscular manifestations and thus confirm that these patients have GSD IIIb. The homozygous IVS32-12A>G mutation found in 4 families segregating GSD IIIb has been previously reported in the Japanese population.3 The A to G transition mutation (IVS32-12A>G) found in our families gave rise to a new 3′ splice site, and an 11-bp intronic sequence was inserted between exons 32 and 33 in the AGL mRNA (Figure 3). This is predicted to result in a truncated enzyme with the loss of 112 carboxyl-terminal amino acids due to premature termination. Genotyping with microsatellite markers and haplotype construction demonstrated that the splice acceptor site mutation IVS32-12A>G in 4 families (A, C, D, and E) appeared on very similar haplotypes, suggesting that the mutation in these 4 families was due to a single mutation event. This is the first report of a mutation in GSD III patients from Saudi Arabia. The mutation was not detected in 70 control chromosomes. Nonetheless, with the high rate of consanguinity in Saudi Arabia, screening for this mutation can improve the molecular diagnosis of GSD III in the relatives of these and other GSD III families. It was intriguing that in family B, the sequence analysis failed to detect any pathogenic variants in the entire AGL gene that could be responsible for the GSD III phenotype. The haplotype analysis showed heterozygous alleles for all 4 microsatellite markers surrounding the AGL gene, suggesting that the phenotype in family B is not linked with the AGL gene, thus ruling out the involvement of the AGL gene in the disease pathogenesis in family B. Several mutations in the AGL gene have been identified so far.14–18 It has been postulated that the mutations in exon 3 can give rise to GSD IIIb, and that GSD IIIa could be distinguished from GSD IIIb by analysis of exon 3.1. Our report, and the previous report of Okubu et al3 provide evidence that mutations in exons other than exon 3 could be responsible for the GSD IIIb phenotype. In summary, we have identified the molecular basis of GSD III in 4 Saudi families without liver or muscle biopsies. The mutation identified will be useful for carrier detection and prenatal diagnosis of the disease. Acknowledgments We thank Dr Mohammed Hannan for providing scientific advice and for the critical reading of this manuscript. We acknowledge Dr Khalid M Al-Harbi for encouraging the conduct of this study. We are also thankful to the patients and their families for their cooperation. This project was supported by the Deanship of Scientific Research, Taibah University, under grant number 6195. Figure 1 Pedigrees of five Saudi families segregating GSD III. Double lines are indicative of consanguineous union. Clear symbols represent unaffected individuals while filled symbols represent affected individuals. Symbols with asterisks indicate individuals who were clinically examined and for whom DNA samples were available for molecular analysis. Figure 2 Sequence analysis of the splice site mutation identified in 4 families (A, C, D, E). The upper panel (A) represents the nucleotide sequences in the affected individuals, the middle panel (B) in the heterozygous carriers and the lower panel (C) in the affected individuals. Arrows in the panels indicate the position of the nucleotide change. Figure 3 Sequence analysis of the splice site mutation using mRNA as a template. The upper panel (A) shows sequence chromatogram obtained by sequencing cDNA of the AGL gene. 11-bps insertion is underlined. The lower panel (B) shows cDNA sequence of the patient aligned with reference sequence. Table 1 Clinical and biochemical features of GSD III patients. Family Number Age/Gender Disease onset Hypoglycemia Hepatomegaly Transaminases ALT/AST CK A, II: 1 5 Yr/M 3 Yr Yes Yes 99/108 834 A, II: 3 9 Yr/F 1 Yr Yes Yes 463/359 436 A, II: 4 11 Yr/F 3 Yr Yes Yes 54/78 – B, II: 3 12 Yr/M 9 Mo No Yes 141/104 – B, II: 4 8 Yr/M 9 Mo No Yes 202/152 232 C, IV: 3 2 Yr/F 1 Yr No Yes 355/487 61 D, IV: 2 36 Yr/F 2 Yr Yes Yes – – D, IV: 3 42 Yr/F 3 Yr Yes Yes 768/877 3,120 D, IV: 4 44 Yr/M 2 Yr Yes Yes – – D, IV: 7 44 Yr/F 3 Yr Yes Yes – – D, IV: 8 46 Yr/F 2 Yr Yes Yes – – D, IV: 9 50 Yr/M 4 Yr Yes Yes 657/766 3,420 D, V: 1 11 Yr/F 2 Yr Yes Yes 212/190 920 E, III: 1 4 Yr/F 1 Yr Yes Yes – – E, III: 5 11 Yr/F 2 Yr Yes Yes 212/190 920 Notes: Five consanguineous Saudi families (A, B, C, D, and E) originating from Madinah in western Saudi Arabia were investigated. Abbreviations: GSD III: glycogen storage disease type 3; ALT: alanine transaminase; AST: aspartate transaminase; CK: creatine kinase; M: male; Yr: years; M: male; F: female; Mo: months. ==== Refs REFERENCES 1 Shen J Bao Y Liu HM Lee P Leonard JV Chen YT 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 1996 98 2 352 357 8755644 2 Van Hoof F Hers HG The subgroups of type 3 glycogenosis Eur J Biochem 1967 2 3 265 270 5235982 3 Okubo M Horinishi A Nakamura N 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 1998 102 1 1 5 9490286 4 Lucchiari S Pagliarani S Salani S Hepatic and neuromuscular forms of glycogenosis type III: nine mutations in AGL Hum Mutat 2006 27 6 600 601 5 Hershkovitz E Forschner I Mandel H Glycogen storage disease type III in Israel: presentation and long-term outcome Pediatr Endocrinol Rev 2014 11 3 318 323 24716397 6 Ismail H Glycogen storage disease type III presenting with secondary diabetes and managed with insulin: a case report Cases J 2009 2 6891 19829878 7 Shen JJ Chen YT Molecular characterization of glycogen storage disease type III Curr Mol Med 2002 2 2 167 175 11949933 8 Liu KM Wu JY Chen YT Mouse model of glycogen storage disease type III Mol Genet Metab 2014 111 4 467 476 24613482 9 Okubo M Horinishi A Takeuchi M Heterogeneous mutations in the glycogen-debranching enzyme gene are responsible for glycogen storage disease type IIIa in Japan Hum Genet 2000 106 1 108 115 10982190 10 Shaiu WL Kishnani PS Shen J Liu HM Chen YT Genotype-phenotype correlation in two frequent mutations and mutation update in type III glycogen storage disease Mol Genet Metab 2000 69 1 16 23 10655153 11 Parvari R Moses S Shen J Hershkovitz E Lerner A Chen YT 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 1997 5 5 266 270 9412782 12 Aldahmesh MA Abu-Safieh L Khan AO Allelic heterogeneity in inbred populations: the Saudi experience with Alström syndrome as an illustrative example Am J Med Genet A 2009 149A 4 662 665 19283855 13 Basit S Ali G Wasif N Ansar M Ahmad W Genetic mapping of a novel hypotrichosis locus to chromosome 7p21.3-p22.3 in a Pakistani family and screening of the candidate genes Hum Genet 2010 128 2 213 220 20544222 14 Ko JS Moon JS Seo JK Yang HR Chang JY Park SS A mutation analysis of the AGL gene in Korean patients with glycogen storage disease type III J Hum Genet 2014 59 1 42 45 24257475 15 Sentner CP Vos YJ Niezen-Koning KN Mol B Smit GP Mutation analysis in glycogen storage disease type III patients in the Netherlands: novel genotype-phenotype relationships and five novel mutations in the AGL gene JIMD Rep 2013 7 19 26 23430490 16 Wu XY Pan JX Guo YB AGL gene analysis of a pedigree with glycogen storage disease type III and identification of a novel mutation Zhonghua Er Ke Za Zhi 2013 51 12 915 919 [Chinese] 24495762 17 Cherif W Ben Rhouma F Messai H High frequency of W1327X mutation in glycogen storage disease type III patients from central Tunisia Ann Biol Clin (Paris) 2012 70 6 648 650 23207808 18 Mili A Ben Charfeddine I Mamaï O Molecular and biochemical characterization of Tunisian patients with glycogen storage disease type III J Hum Genet 2012 57 3 170 175 22089644