Sultan Qaboos University Med J, November 2013, Vol. 13, Iss. 4, pp. 502-509, Epub. 8th Oct 13 Submitted 18TH Nov 12 Revisions Req. 29TH Jan & 1ST May 13; Revisions Recd. 1ST Apr & 8TH Jul 13 Accepted 1ST Aug 13 1Department of Biology, College of Science; Departments of 2Pathology and 3Genetics, Sultan Qaboos University Hospital; 4Department of Pathology, College of Medicine & Health Sciences, Sultan Qaboos University, Muscat, Oman *Corresponding Author e-mail: alansari@squ.edu.om حتديد الطفرات املسببة ملرض بوميب يف عينات أنسجة حمفوظة وتطوير الفحص اجلزيئي علياء الأن�سارية، �سمرية الرواحية، طاهر باعمر، مرمي النبهانية، اأناند داتي مر�ض بومبي )مر�ض تخزين الغليكوجني من النوع الثاين( هو مر�ض �سبغى ج�سدي متنحي لالختزان يف اجل�سيمات امللخ�ص: الهدف: بهذا امل�سابني الطفال يفيد ان ميكن الأنزميية بال�ستعا�سة للعالج املبكر ال�ستخدام غلوكوزيداز. األفا حم�ض نق�ض عن ينتج احلالة الأن�سجة من النووي احلم�ض ا�ستخراج مت الدرا�سة، هذه يف ال�رسيرية. املظاهر وتغايرية املر�ض ندرة الت�سخي�ض يعوق ولكن املر�ض الأر�سيفية )FFPET( لتحديد الطفرات امل�سببة ملر�ض بومبي يف عمان و عمل اختبار جزيئي. الطريقة: مت ت�سميم امل�رسعات الإنرتونية مل�ساعفة مقاطع ق�سرية )454-193 قاعدة مزدوجة( من املحورات 20-2( للبحث عن الطفرات با�ستخدام الت�سل�سل املبا�رس. النتائج: مت التعرف على طفرتني من الطفرات املت�سببة للمر�ض الوخيم يف عينتني. الأوىل برمز طفرة p.Arg854X( c.2560CT (p.Arg854X), and the second was found at a splice acceptor site, c.1327-2A>G. Polymerase chain reaction- and restriction fragment length polymorphism-based tests were designed for the rapid genotyping of the identified mutations. Conclusion: These tests can facilitate prenatal diagnosis and help in identifying carriers in families with the identified mutations. Keywords: Pompe Disease; Glucan 1,4-alpha-Glucosidase; Tissue; Mutations; Genotyping Techniques; Oman. The Identification of Pompe Disease Mutations in Archival Tissues and Development of a Rapid Molecular-based Test *Aliya Alansari,1 Samira Al-Rawahi,2 Taher Ba-Omar,1 Mariam Al-Nabhani,3 Anand Date4 clinical & Basic research Advances in Knowledge - This study identified two genetic mutations causing Pompe disease in two Omani patients. Application to Patient Care - The molecular test designed in this study can facilitate prenatal diagnosis and the screening of at-risk infants. Pompe disease is a rare autosomal recessive disease, classified as a lysosomal storage disorder. It is caused by an acid alpha-glucosidase (GAA EC. 3.2.1.20) deficiency, which results in the accumulation of glycogen within the lysosomes and the cytoplasm of the cardiac, skeletal and smooth muscle cells. The clinical spectrum of the disease varies in terms of the age of onset, the disease progression rate and the extent of organ involvement.1,2 Because of the continuous clinical spectrum of Pompe disease, Gungor and Reuser proposed three Identification of Pompe Disease Mutations in Archival Tissues and Development of a Rapid Molecular-based Test 503 | SQU Medical Journal, November 2013, Volume 13, Issue 4 subtypes: classic infantile, childhood and adult.3 The first is characterised by an onset of symptoms within the first year of life, which is always associated with hypertrophic cardiomyopathy and a total lack of acid α-glucosidase activity; the second subtype covers patients with an onset of symptoms between birth and adolescence, but without persistent and/or progressive cardiac hypertrophy, and the third subtype covers patients with an onset of symptoms between adolescence and late adulthood.3,4 Early diagnosis and enzyme-replacement therapy can benefit infants with Pompe disease.5 To establish a diagnosis of Pompe disease, both clinical evaluation and diagnostic tests are required.1 Rapid blood-based activity tests were developed for pre-symptomatic diagnoses at birth and for at-risk individuals, to allow optimal conditions for enzyme replacement therapy;6 however, a second confirmatory test is recommended to support the diagnoses. DNA-based testing is an approach that can rapidly confirm the diagnosis and identify the nature of the mutations (genotypes) that alter the level of residual enzyme activity and are responsible for the clinical phenotype heterogeneity.7,8 The GAA gene is about 28 Kilo-base pairs (Kb) long, contains 20 exons and maps to human chromosome 17q25.2–q25.3. The first exon is non-coding and the complementary DNA (cDNA) encodes a protein of 952 amino acids.2 Nearly 250 mutations have been identified as causing Pompe disease, and they are rated by severity and divided into different classes.9 Some of these mutations are common in certain populations.2 The identification of mutations in a population overcomes the problem of a lengthy, time- consuming and expensive search for a mutation approach—due to the size of the gene—by providing a limited number of mutations to be tested. This in turn facilitates diagnoses and aids in the counselling of patients and families with the disease. In this study, we utilised archived formalin-fixed paraffin embedded (FFPE) tissues in pathology laboratories to identify retrospectively GAA mutations causing Pompe disease (glycogen storage disease type II) in Oman, which enabled the design of a rapid molecular test for families with the identified mutations. Methods Archived muscle tissues were collected in 2000 and 2002 from two infants of Arab origin (a 3-month- old male and a 4-month-old female, respectively) with clinical presentations consistent with infantile onset Pompe disease. Both infants had presented with bronchitis, hypotonia and hypertrophic cardiomyopathy. Their creatine kinase (CK) levels were high at 571 u/L and 410 u/L, respectively (range 0–6 u/L), and lactate dehydrogenase (LDH) levels were high at 2,415 u/L and 626 u/L, respectively (range 91–180 u/L). The electromyograms (EMG) showed mixed myopathic and neuropathic pictures. For the DNA extraction, 10 sections of 7 micrometres (μm) were collected from archived muscle biopsies for each infant and kept in 2 ml microcentrifuge tubes. Normal archived muscle tissue sections were used as a control. DNA extraction was performed using a modified phenol- chloroform extraction method.10 The sections were deparaffinised twice in 1 ml of pre-heated xylene (for 5 mins each) and then re-hydrated with 1 ml of 99% ethanol and 95% ethanol (twice, for 3 mins). For digestion, the tissues were incubated overnight at 60 ºC with 300 µl of lysis buffer solution containing 100 µl of 5 mg/ml proteinase K (the proteinase K was inactivated at 95 ºC for 15 mins). To purify the DNA, the samples were extracted twice with 300 µl of phenol and once with 350 µl of chloroform- isoamyl alcohol (at a ratio of 24:1). Finally, the DNA was precipitated with one ml of 100% ethanol and one U/L of glycogen (20 mg/ml). The mix was incubated at -20 ºC for one hr and then centrifuged at 4 ºC for 15 mins at 13,000 rpm. After washing the pellet with 1 ml of 70% ethanol, the extracted DNA was left to air-dry and was then resuspended in 50 µl of double-distilled water (ddH2O). The integrity of the DNA was checked using 1% agarose gel electrophoresis and the quality of the DNA was analysed by polymerase chain reaction (PCR) for different primer sets that amplify different fragment sizes (250–500 bp). For the PCR and sequencing process, 17 sets of intronic primers flanking exons were designed to amplify the coding regions [Table 1]. Exon 2 is a large exon and was therefore amplified with two overlapping fragments. The primers were designed to amplify short fragments (193–454 bp) suitable for degraded DNA.11 PCR was performed using a 25 µl Aliya Alansari, Samira Al-Rawahi, Taher Ba-Omar, Mariam Al-Nabhani and Anand Date Clinical and Basic Research | 504 reaction volume containing the PCR buffer, 5 µl of genomic DNA, 1.5 millimolars (mM) of magnesium chloride (MgCl2), 0.5 unified atomic mass units (U) of Taq DNA polymerase (Promega Corporation, Madison, Wisconsin, USA), 200 micromolars (µM) of deoxyribonucleotide triphosphates (dNTP) and 0.5 µM of each primer. The PCR was performed under the following conditions: 95 °C for 5 mins followed by 35 cycles of denaturation at 95 °C for 30 secs, annealing at 58– 63 °C for 30 secs and then extension at 72 °C for 30 secs. A final extension step was carried out at 72 °C for 5 mins. The DNA isolated from the peripheral blood was used as positive controls and ddH2O was used as a negative control. The product was checked for specificity and yield using 2% agarose gel stained with ethidium bromide. The direct sequencing (DS) of the PCR products was performed by a commercial company (Macrogen, Inc., Seoul, Korea). Chromatograms were edited and analysed using the software BioEdit, Version 6.0.7 (Ibis Biosciences, Carlsbad, California, USA). Regarding the restriction fragment polymorphism (RFLP), the mutation at codon 854 in exon 18, c.2560C>T, correlated with a Bsu36I restriction site in the T allele. For the restriction fragment length polymorphism (RFLP) reaction, 7 µl of the exon 18 PCR product was digested for 4 hrs with 10 U of the Bsu36I gene (New England BioLabs, (UK) Ltd., Hitchin, Herts, UK ) at 37 °C. After the inactivation of the enzyme at 80 °C for 30 mins, the restriction fragments were separated by electrophoresis using a 2.5% agarose gel. The 250 bp PCR product was cleaved into two fragments of 151 and 99 bp in the T allele and uncleaved in the C allele. The intron 8 splicing site mutation, c.1327- 2A>G, correlated with a SmaI restriction site in the G allele. For the RFLP reaction, 7 µl of exon 7 and 8 of the PCR product was digested for 4 hrs with 10 U of the SmaI (New England BioLabs) at 37 °C. After the inactivation of the enzyme at 80 °C for 30 mins, the restriction fragments were separated by electrophoresis using a 2.5% agarose gel. The 252 bp PCR product was cleaved into two fragments of 162 and 90 bp in the G allele and uncleaved in the A allele. The project was approved by the Ethics Research Committee of the College of Medicine & Health Sciences at Sultan Qaboos University in Muscat, Oman. Results The examination of the muscle biopsies by light microscopy and electron microscopy for both patients showed vacuolar myopathy with storage of glycogen consistent with Pompe disease [Figures 1 and 2]. Table 1: Primer sequences, fragment sizes and annealing temperatures Exon primers Sequence (5' to 3') Fragment size (bp) Annealing temp. ˚C 2 A F ctgagcccgctttcttctc 365 61 2 A R cactgttcctgggtgatgg 365 61 2 B F ccatcacccaggaacagtg 330 61 2 B R gtgaggtgcgtgggtgtc 330 61 3 F ggtggctgtggggaacat 210 59 3 R ctggcacagagcccagaa 210 59 4 + 5 F ggtgctctcaggctcgtgt 454 59 4 + 5 R aggcagcacggaggagac 454 59 6 F tgaagaatctgtcccccaac 342 61 6 R ggtcatgttctccaccacct 342 61 7 + 8 F ctcatgaagtcggcgttgg 400 61.5 7 + 8 R ccttcccaaagacccacagt 400 61.5 9 F cccagcctcatcctctcact 252 63 9R gctggaggcctctgctttct 252 63 10 + 11 F cactgcagcctctcgttgtc 396 58 10 + 11 R gctaagtctcccaggccaga 396 58 12 F gaggaagctccctggaaacc 202 59 12 R acaggctgtgagggcagag 202 59 13 F ctctgcctcatcccagaaag 287 63 13 R cccggctctactctgctg 287 63 14 F cctgaggaccagcctgact 248 59 14 R attcccaggggagagtcttg 248 59 15 F agcacccaagtgcttcctt 209 61.5 15 R gcccctgcctaggtcact 209 61.5 16 F gtatgcctgtgtgcccatc 193 59 16 R ggcttagggtccccagact 193 59 17 F cccagaatcctcaaagcaac 232 61.5 17 R ctctgcagtgtgctgtccac 232 61.5 18 F cacgtgtccttccctttcc 250 61.5 18 R ccctcaccccttctcaacc 250 61.5 19 F ctgtctgctgacacctccac 239 61.5 19 R cgatccctgtgccactct 239 61.5 20 F tgctccatttgtgctctctc 200 61.5 20 R ctccaggtgacacatgcaac 200 61.5 bp = base pairs; temp. = temperature. Identification of Pompe Disease Mutations in Archival Tissues and Development of a Rapid Molecular-based Test 505 | SQU Medical Journal, November 2013, Volume 13, Issue 4 DNA was successfully extracted from the two archived tissues from the patients with Pompe disease using a modified phenol-chloroform extraction method. The checking of the integrity and quality of the DNA by agarose gel electrophoresis and PCR, respectively, indicated a good DNA quality for PCR and post-PCR methods. Two mutations were identified in the Pompe disease samples. Infant 1 (female) was homozygous for mutation c.2560C>T (p.Arg854X) [Figure 3] and infant 2 (male) was homozygous for mutation c.1327-2A>G [Figure 4]. Neither of the two identified mutations were found in the DNA control sample, nor was the sequence retrieved from the GenBank sequence database (NW_926918),12 but both were found in the Pompe Center database.13 The PCR and RFLP tests for both mutations were designed to be used as fast confirmatory tests Figure 1 A to F. Micrographs of infant 1. A & B: Light micrographs showing vacuolated fibres (arrows) stained with haematoxylin and eosin and Masson’s trichrome. C: Light micrograph showing the rich glycogen accumulation (arrows) with Periodic acid-Schiff (PAS) stain. D: Light micrograph showing the digested glycogen with a diastase (arrow) stained with PAS diastase. E: Light micrograph showing type I (arrows) and type II (arrows) fibres stained with adenosine triphosphatase (pH = 9.5). F: Electron micrograph showing lysosomal vacuoles containing small aggregates of glycogen (arrows). Scale is 200 µm for A–E and 500 nm for F. Aliya Alansari, Samira Al-Rawahi, Taher Ba-Omar, Mariam Al-Nabhani and Anand Date Clinical and Basic Research | 506 for diagnosis and screening. Typical genotyping results are shown in Figures 5 and 6. Discussion Muscle biopsies have always been considered the standard method for diagnosing Pompe disease.4 However, biopsies are invasive methods requiring anaesthesia, which is not recommended for infants with Pompe disease. In 2006, enzyme replacement therapy became available and, to ensure the patient’s optimal benefit, new rapid and less invasive diagnostic tests were developed.4 Testing GAA in dried blood is one of the methods developed and it is considered to be reliable and suitable for newborn screening, as it only requires a drop of blood to be collected, either from the heel or using the finger stick method.6 However, the Pompe disease Figure 2 A to F. Micrographs of infant 2. A & B: Light micrographs showing the funicular architecture of the tissue; there are many vacuoles of variable sizes within the fibres (arrows) stained with haematoxylin and eosin and Masson’s trichrome. C: Light micrograph showing the glycogen accumulation (arrows) with Periodic acid-Schiff (PAS) stain. D: Light micrograph showing the digested glycogen with a diastase (arrows) stained with PAS diastase. E: Light micrograph showing type I and II fibres (arrows) stained with nicotinamide adenine dinucleotide-tetrazolium reductase. F: Electron micrograph showing lysosomal vacuoles containing aggregated granules of glycogen (arrows). Scale is 200 µm for A–E and 500 nm for F. Identification of Pompe Disease Mutations in Archival Tissues and Development of a Rapid Molecular-based Test 507 | SQU Medical Journal, November 2013, Volume 13, Issue 4 diagnostic working group emphasised the need for a second confirmatory test to support the clinical and/or biochemical diagnosis due to the absence of developed quality assurance measures.4 The detection of Pompe disease-causing mutation(s) is considered a definitive and rapid confirmatory test. It can be utilised in screening at-risk infants and for the identification of carriers, as well as in prenatal diagnosis and the genetic counselling of families with a history of Pompe disease. In this retrospective study, the pathology archival formalin-fixed paraffin-embedded (FFPE) tissues provided a source of good quality DNA to investigate the molecular bases of Pompe disease in two Omani infants. Optimisation of the DNA extraction method was very important and the primers for PCR were designed to amplify short fragments (less than 450 bp). The analysis of exons 2–20 of the GAA gene using direct sequencing identified two mutations. In infant 1, a c.2560C>T (p.Arg854X) mutation in a CpG dinucleotide, which is susceptible to recurrent mutations, was identified. It is a transition, nonsense mutation at the C-terminal end that is known to be associated with infantile onset Pompe disease.8 Although found among Pakistani, Mexican- American and French ethnicities, the mutation has been observed in up to 60% of patients of African descent with a common haplotype.2 In infant 2, a c.1327-2A>G mutation modifying the splice acceptor site of exon 9 was identified. This mutation is associated with very severe effects, and has been reported in Pompe disease cases from UK, Iran and in patients of Arab origin.14 The presence of both mutations as a homozygous genotype is an indicator of parental consanguinity, which has been reported in more Omani patients with inborn errors of metabolism (81%) compared to the general population (33%).15 Furthermore, consanguineous marriage in Oman (52%) has been found to be dominated by first-cousin marriages (75%).16 Conclusion This study provided valuable information regarding Pompe disease-causing mutations in the GAA gene and has enabled the development of specific molecular tests for Omani families. An RFLP test was developed to identify each mutation. The timescale for carrying out the PCR and RFLP test on a routine basis is about 3–8 hrs and is therefore fast compared to other available methods. To the best of our knowledge, this is the first retrospective study to utilise FFPE tissues in Oman to identify the mutations causing Pompe disease. We were successful in identifying two Pompe disease-causing mutations and developing a rapid specific genetic test that can be used for prenatal Figure 3 Panels A & B. The c.2560C>T (p.Arg854X) mutation in exon 18. A: The alignment of the wild type and mutant sequences. B: Direct sequencing chromatograms for the homozygous wild type (Cont.) and mutation (infant 1) sequences. Figure 4 Panels A & B. The c.1327-2A>G mutation in intron 8. A: The alignment of the wild type and mutant sequences. B: Direct sequencing chromatograms for the homozygous wild type (Cont.) and mutation (infant 2) sequences. Aliya Alansari, Samira Al-Rawahi, Taher Ba-Omar, Mariam Al-Nabhani and Anand Date Clinical and Basic Research | 508 diagnosis and carrier screening in Omani families with the identified mutations. References 1. ACMG Work Group on Management of Pompe Disease: Kishnani PS, Steiner RD, Bali D, Berger K, Byrne BJ, et al. Pompe disease diagnosis and management guideline. Genet Med 2006; 8:267–88. 2. Raben N, Plotz P, Byrne BJ. Acid alpha-glucosidase deficiency (glycogenosis type II, Pompe disease). Curr Mol Med 2002; 2:145–66. 3. Güngör D, Reuser AJ. How to describe the clinical spectrum in Pompe disease? Am J Med Genet 2013; 161A:399–400. 4. Beckemeyer AA, Mendelsohn NJ, Kishnani PS. Response to the letter “How to describe the clinical spectrum in Pompe disease?”. Am J Med Genet A 2013; 161A:401–2. 5. Chien YH, Lee NC, Thurberg BL, Chiang SC, Zhang XK, Keutzer J, et al. Pompe Disease in infants: Improving the prognosis by newborn screening and early treatment. Pediatrics 2009; 124:e1116–25. 6. Gasparotto N, Tomanin R, Frigo AC, Niizawa G, Pasquini MB, Blanco M, et al. Rapid diagnostic testing procedures for lysosomal storage disorders: Alpha-glucosidase and beta-galactosidase assays on dried blood spots. Clin Chim Acta 2009; 402:38–41. 7. Kroos MA, Van der Kraan M, Van Diggelen OP, Kleijer WJ, Reuser AJ, Van den Boogaard MJ, et al. Glycogen storage disease type II: Frequency of three common mutant alleles and their associated clinical phenotypes studied in 121 patients. J Med Genet 1995; 32:836–7. 8. Hermans MM, van Leenen D, Kroos MA, Beesley CE, Van Der Ploeg AT, Sakuraba H, et al. Twenty-two novel mutations in the lysosomal alpha-glucosidase gene (GAA) underscore the genotype-phenotype correlation in glycogen storage disease type II. Hum Mutat 2004; 23:47–56. 9. Kroos M, Hoogeveen-Westerveld M, Michelakakis H, Pomponio R, Van der Ploeg A, Halley D, et al. Update of the pompe disease mutation database with 60 novel GAA sequence variants and additional studies on the functional effect of 34 previously reported variants. Hum Mutat 2012; 33:1161–5. 10. Cao W, Hashibe M, Rao JY, Morgenstern H, Zhang ZF. Comparison of methods for DNA extraction from paraffin-embedded tissues and buccal cells. Cancer Detec Prev 2003; 27:397–404. 11. Libório TN, Etges A, da Costa Neves A, Mesquita RA, Nunes FD. Evaluation of the genomic DNA extracted from formalin-fixed, paraffin-embedded oral samples archived for the past 40-years. J Bras Patol Med Lab 2005; 41:405-10. 12. National Center for Biotechnology Information. GenBank Overview. Available at: http://www.ncbi. nlm.nih.gov/genbank/ Accessed: Aug 2013. 13. Erasmus University. Pompe Center. Available at: http://w w w.erasmusmc .nl/klinische_genetica/ research/lijnen/pompe_center/?lang=en Accessed: Aug 2013. Figure 5 Panels A & B. The assay design (A) and results (B) of the polymerase chain reaction restriction fragment length polymorphism analysis for the c.2560C>T (p.Arg854X) mutation in exon 18. A: First, a 250 bp fragment of the GAA gene covering the mutation was amplified by polymerase chain reaction (PCR); next, the PCR product was digested with the Bsu36I restriction enzyme, which recognizes only the T allele, and generated two fragments, 151 bp and 99 bp. B: The electrophoretic separation of the Bsu36I enzymatic restriction products using 2.5% (weight/volume percentage) agarose gel. Lanes: 1 = DNA ladder; 2 = TT homozygote (mutant type) digested product; 3 = CC homozygote (wild type) undigested fragment (250 bp), and 4 = CC homozygote (wild type) undigested fragment (250 bp). Figure 6 Panels A & B. The assay design (A) and results (B) of the polymerase chain reaction restriction fragment length polymorphism analysis for the c.1327-2A>G mutation in intron 8. A: First, a 252 bp fragment of the GAA gene covering the mutation was amplified by polymerase chain reaction (PCR); next, the PCR product was digested with the SmaI restriction enzyme which recognizes only the G allele, and generated two fragments, 162 bp and 90 bp. B: The electrophoretic separation of the SmaI enzymatic restriction products using 2.5% (weight/volume percentage) agarose gel. Lanes: 1 = DNA ladder; 2 = GG homozygote (mutant type) digested product; 3 = GG homozygote (mutant type) digested product, and 4 = AA homozygote (wild type) undigested fragment (252 bp). Identification of Pompe Disease Mutations in Archival Tissues and Development of a Rapid Molecular-based Test 509 | SQU Medical Journal, November 2013, Volume 13, Issue 4 14. Kroos M, Pomponio RJ, van Vliet L, Palmer RE, Phipps M, Van der Helm R, et al. Update of the Pompe disease mutation database with 107 sequence variants and a format for severity rating. Hum Mutat 2008; 29:E13–26. 15. Joshi SN, Venugopalan P. Clinical characteristics of neonates with inborn errors of metabolism detected by Tandem MS analysis in Oman. Brain Dev 2007; 29:543–6. 16. Islam MM, Dorvlo AS, Al-Qasmi AM. The pattern of female nuptiality in Oman. Sultan Qaboos Univ Med J 2013; 13:32–42.