- Email: [email protected]
Glycogen storage disease type II in Spanish patients: High frequency of c.1076-1G>C mutation
Molecular Genetics and Metabolism 92 (2007) 183–187 www.elsevier.com/locate/ymgme
Brief Communication
Glycogen storage disease type II in Spanish patients: High frequency of c.1076-1G>C mutation Laura Gort, M. Josep Coll, Amparo Chaba´s
*
Institut de Bioquı´mica Clı´nica, Hospital Clı´nic, C/ Mejı´a Lequerica s/n, Edifici Helios III, 08028 Barcelona, Spain Centro de Investigacio´n Biome´dica de Enfermedades Raras (CIBERER), Barcelona, Spain Received 9 March 2007; received in revised form 24 May 2007; accepted 24 May 2007 Available online 5 July 2007
Abstract Glycogen storage disease type II is an autosomal recessive disorder of glycogen metabolism due to deficiency of lysosomal acid a-glucosidase. We present the molecular and enzymatic analyses of 22 Spanish GSD II patients. Molecular analyses revealed nine novel mutations. The most common defects were mutations c.-32-13T>G (25%) and c.1076-1G>C (14%) and we report the first homozygous patient for c.1076-1G>C mutation presenting with an infantile form. Alleles bearing mutation c.-32-13T>G are associated with the same haplotype. 2007 Elsevier Inc. All rights reserved. Keywords: Acid a-glucosidase; GAA; Glycogen storage disease; Haplotype analysis; Lysosomal storage disorder; Molecular analysis; Pompe disease
Introduction Glycogen storage disease type II (GSD II, Pompe disease, MIM 232300) is an autosomal recessive disorder of glycogen metabolism due to deficiency of lysosomal acid a-glucosidase (GAA, acid maltase, EC 3.2.1.3). Accumulation of glycogen in the lysosomal apparatus leads to infantile-onset and late-onset (juvenile and adult) forms, but there is a continuum of clinical phenotypes. The infantileonset form is characterized by severe cardiomyopathy, cardiomegaly, profound muscle weakness, delayed motor development and progressive respiratory insufficiency. The juvenile and adult-onset forms present with slowly progressive myopathy and generally without cardiac involvement. A broad spectrum of pathogenic mutations and polymorphic changes has been identified in the lysosomal acid a-glucosidase gene (GAA) (www.pompecenter.nl). Many mutations were identified only once,
although some are common to specific ethnic groups [1– 3]. Mutation c.-32-13T>G is frequent among Caucasian patients presenting late-onset forms [4,5]. Here we present the molecular analysis and enzymatic activity levels of 22 Spanish GSD II patients with identification of 23 mutations, nine of them new. The frequency of the severe mutation c.1076-1G>C is relatively high in both infantile- and late-onset patients. Material and methods Patients Patients were unrelated and came from different regions of Spain. Clinical information was provided by the patient’s physician and diagnosis of GSD II was confirmed by enzymatic determination of GAA activity, except for patient #19 in whom disease was suspected after histological examination of a muscle biopsy and later confirmed by mutation analysis of the GAA gene.
Biochemical assays * Corresponding author. Address: Institut de Bioquı´mica Clı´nica, Hospital Clı´nic, C/ Mejı´a Lequerica s/n, Edifici Helios III, 08028 Barcelona, Spain. Fax: +34 93 227 5668. E-mail address: [email protected] (A. Chaba´s).
1096-7192/$ - see front matter 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.ymgme.2007.05.011
GAA activity was measured in cultured skin fibroblasts and isolated lymphocytes, because of the interfering maltase–glucoamylase activity present in neutrophils, with the fluorogenic substrate 4-methylumbelliferyl-a-D-glucopyranoside [6].
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L. Gort et al. / Molecular Genetics and Metabolism 92 (2007) 183–187
Molecular analysis
Discussion
Genomic DNA was extracted from whole blood or fibroblasts using standard protocols. Each exon and intron boundaries of the GAA gene was PCR-amplified using self-designed oligonucleotides and standard protocols. PCR products were directly sequenced. Gene nucleotide numbering is according to sequence RefSeq NM_000152, with +1 as A of the ATG start codon. The ATG codon represents +1 for the amino acid numbering according to a-glucosidase preprotein sequence NP_000143.
The most frequent mutation in Spanish patients is the splicing mutation c.-32-13T>G (total allele frequency 25% and 34.4% of late-onset alleles) which has been reported to be the most common among the Caucasian late-onset affected population [4]. This frequency is low when compared to previous reports: 68% [4] and 42.3% [7]. In our study all the patients carrying this mutation also presented with the juvenile or adult form of the disease, corroborating the suggestion that this mutation is a mild one and that clinical manifestations depend upon the severity of the mutation in the other allele [7]. Within a clinical phenotype, patients bearing this mutation, even when associated with the same haplotype, may manifest initial symptoms at different ages and may present different disease severity, indicating that secondary factors may influence the clinical course [8–13]. The second most frequent mutation in the Spanish GSDII population is the splicing change c.1076-1G>C, with allele frequency of 14%; this mutation has not been previously detected in larger series of patients except at heterozygosity in one patient and it is presumed to be causative of an insertion (r.1076-79_1195+89ins) [7]. In the Spanish series mutation c.1076-1G>C was found at homozygosis in an infantile patient, with severe cardiomyopathy, cardiomegaly and hepatomegaly. This would indicate that mutation c.1076-1G>C is severe, and the fact that patients carrying this mutation present with the juvenile or adult form of the disease may be due to the mildness of the mutation on the other allele. Other novel mutations have been found at heterozygosity only in one or two patients so that expression studies are necessary to establish the severity of these changes. Among previously reported mutations, mutation c.546G>C is interesting. Although it does not change the amino acid in position 182 (Thr), it affects the last nucleotide of exon 2 and it is presumed to cause leaky splicing [14]. All 11 alleles bearing mutation c.-32-13T>G in Spanish patients are associated with the same haplotype, the DHRGEVVT haplotype as named after Kroos et al. [8], and we have extended this haplotype with 12 more informative polymorphic markers. Although this mutation has occurred more than once on different haplotypes, the presence of a single haplotype associated with this change in Spain suggest a possible founder effect. Mutation c.10761G>C was carried by six alleles (five patients) but only in one case this mutation was associated with the same haplotype as mutation c.-32-13T>G, while in five alleles mutation is associated with a different haplotype. This last haplotype is based on the first most common GAA core haplotype (DRHGEIVT according to [8]), except for position E (c.2065G) that in our five alleles is K (c.2065A). These results would suggest that this frequent mutation among Spanish patients occurred at least twice although most patients originate from the same region.
Results Biochemical findings Some correlation was found between fibroblast residual GAA activity and clinical phenotype. Mean ± SD GAA activity levels were 2.0 ± 0.9%, 7.1 ± 5.4% and 20.2 ± 7.7% of mean control values in the infantile, juvenile and adult patients, respectively, although the level of residual activity ranged widely even in patients with the same disease phenotype. In contrast, in lymphocytes, residual activity ranged from 1.9% (juvenile patient #24) to normal values (adult patient #34). GAA activity, genotypes and phenotypes of the 22 patients are summarized in Table 1. Mutation analysis We analysed 22 unrelated GSDII patients to determine the molecular defect in the GAA gene. We found nine novel and fourteen already described mutations in these patients. The novel mutations are p.Q115X (c.343C>T), p.L169P (c.506T>C), p.L398fsX107 (c.1192dupC), p.Y455C (c.1364A>C), p.P482L (c.1445C>T), p.A644P (c.1930G> C), p.D645Y (c.1933G>T), p.I752del (c.2255_2257delTCA) and p.E867fsX19 (c.2600_2604delTGCTGinsA). None of the missense mutations were found in 100 control chromosomes. The most common defects were c.-3213T>G and c.1076-1G>C mutations accounting for 25% and 14% of total alleles, respectively. Polymorphisms and haplotype analysis Six novel nondeleterious changes in the GAA gene were detected: c.693-49C>T (intron 3); c.852G>A (exon 4); c.1075+13C>T (intron 6); c.2331+151C>T (intron 16) and c.2331+293A>T (intron 16). We also studied associations between mutations c.-32-13T>G and c.1076-1G>C and specific haplotypes (Table 2). All eleven alleles with mutation c.-32-13T>G are presumably associated with the same haplotype. In contrast, for c.1076-1G>C mutation, identified in six alleles, the change was associated with one haplotype in five alleles while the other allele was associated with the same haplotype as mutation c.-32-13T>G. Haplotype association of each mutation was deduced when polymorphic markers revealed homozygosity, and in cases of heterozygosity consistency was checked.
Table 1 Genotype, phenotype and GAA activity in 22 GSD II patients Allele 1
Allele 2
Phenotype
Age at onset
Main clinical traits
Residual GAA activity (%)
2 4 6 7 11 50 5 9 21 24 3 19 22 1 8 12 15 34 47 54
p.L169P c.1076-1G>C c.2481+102_2646+31del p.N470del p.L552P p.Y455C c.-32-13T>G c.-32-13T>G c.-32-13T>G c.-32-13T>G c.1076-1G>C c.1076-1G>C p.P482L c.-32-13T>G c.-32-13T>G c.-32-13T>G c.-32-13T>G c.-32-13T>G c.-32-13T>G c.-32-13T>G
p.D489N c.1076-1G>C c.2646+2T>A p.M408V c.2600_2604delinsA p.G638W p.L552P c.2255_2257delTCA p.G638W p.G309R p.R725W p.R725W p.Q115X p.Y292C p.D645Y c.1192dupC p.R725W p.G478R c.2600_2604delinsA c.546G>C
Infantile Infantile Infantile Infantile Infantile Infantile Juvenile Juvenile Juvenile Juvenile Juvenile Juvenile Juvenile Adult Adult Adult Adult Adult Adult Adult
Birth 4m 4m Birth 6m 2m 6y 3y 5y 2y6m 3y 3y 2y (60y) 30y 31y (44y) NA 35y (65y)
3.0 (F) 3.5 (F) 0.6 (F) 1.8 (F) 2.3 (F) 10 (L);2.4 (F) 4.6 (L) ; 11 (F) 11.4 (F) 9.0 (F) 1.9 (L) 1.1 (F) NA 35 (L) 17 (F) 20 (F) 28 (F) 5.2 (F) 90 (L) 13 (L); 13 (F) 15 (L); 21 (F)
14 17
c.1076-1G>C c.1076-1G>C
p.Q115X p.A644P
Adult Adult
(34y) (34y)
HM, MC H, HM, MD, MC H, HM, MC, leukopenia H, HM, cardiomegaly NA NA H,MD, skeletal abnormalities NA Muscular atrophy, torax deformity H, muscular weakness HM, MD, splenomegaly H, respiratory failure H, MC MD, MC Respiratory muscle weakness Myopathy, muscle weakness MD, vacuolar myopathy NA Strabismus, progressive muscular weakness Chronic pneumonopathy, ischemic cardiopathy, basal dyspnea Muscle weakness, respiratory insufficiency, difficulty feeding H, glaucoma, Marfan syndrome
13 (L) 5 (L)
L. Gort et al. / Molecular Genetics and Metabolism 92 (2007) 183–187
Patient
Gene nucleotide numbering is according to sequence RefSeq NM_000152, with +1 as A of the ATG start codon. Age at diagnosis, in parentheses. NA, not available; H, hypotonia; HM, hepatomegaly; MD, muscular dystrophy; MC, myocardiopathy; F, fibroblasts; L, lymphocytes.
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References
T T C
C C G
A A G
G G A
Ins
T T C
G G A
G G A
A A G
G G C
C C A
G G A
A A G
G G A
T T C
G G A
Ins A A G
The authors thank all the physicians who referred the patients, Drs.: Azan˜a, Barreiro, Castro, Civantos, Colomer, Blanco, Dı´az, Ferna´ndez Villalba, Gala´n, Garcı´a Silva, Gayoso, I. Gonza´lez, N. Gonza´lez, Hermida, Illa, Lo´pez Coronas, Lo´pez de Munain, Lo´pez Va´zquez, Lorente, Martı´nez Garcı´a, Morera, Olive´ Planas, Ortola Devesa, Pascual Pascual, Pe´rez-Sheriff, Rojas, Tamariz-Martel, as well as to A.M. Ban˜on Herna´ndez (Asociacio´n Espan˜ola de Enfermos de Glucogenosis). We thank J. Duque, J. Jarque, H. Selle´s and A. Valle for excellent technical assistance. This study was supported in part by FIS (Redes tema´ticas, G03/054 REDEMETH and 05/1182).
G G A
Acknowledgments
c.-32-13T>G 11 c.1076-1G>C 1 5
c.596 c.668 c.858 c.858 A/G G/A +7ins7 +30T/C Number c.324 c.547-4 of alleles T/C C/G
Table 2 The Spanish most frequent mutations associated haplotypes
c.955 c.1203 c.1327- c.1438- c.1551 c.1888 c.2040 c.2065 c.2133 c.2331 c.2331 c.2338 c.2998insG +12G/A A/G 18A/G 19G/C +49C/A +21G/A +20A/G G/A A/G +20G/A +24T/C G/A
186
[1] M.A. Kroos, M. Van der Kraan, O.P. van Diggelen, W.J. Kleijer, A.J. Reuser, M.J. Van den Boogaard, M.G. Ausems, H.K. Ploos van Amstel, L. Poenaru, M. Nicolino, Glycogen storage disease type II: frequency of three common mutant alleles and their associated clinical phenotypes studied in 121 patients, J. Med. Genet. 32 (1995) 836–837. [2] J.J. Shieh, C.Y. Lin, Frequent mutation in Chinese patients with infantile type of GSD II in Taiwan: evidence for a founder effect, Hum. Mutat. 11 (1998) 306–312. [3] J.A. Becker, J. Vlach, N. Raben, K. Nagaraju, E.M. Adams, M.M. Hermans, A.J.J. Reuser, S.S. Brooks, C.J. Tifft, R. Hirschhorn, M.L. Huie, M. Nicolino, P.H. Plotz, The African origin of the common mutation in African American patients with glycogen-storage disease type II, Am. J. Hum. Genet. 62 (1998) 991–994. [4] M.L. Huie, A.S. Chen, S. Tsujino, S. Shanske, S. DiMauro, A.G. Engel, R. Hirschhorn, Aberrant splicing in adult onset glycogen storage disease type II (GSDII): molecular identification of an IVS1 (-13T fi G) mutation in a majority of patients and a novel IVS10 (+1GT fi CT) mutation, Hum. Mol. Genet. 3 (1994) 2231–2236. [5] C.F. Boerkoel, R. Exelbert, C. Nicastri, R.C. Nichols, F.W. Miller, P.H. Plotz, N. Raben, Leaky splicing mutation in the acid maltase gene is associated with delayed onset of glycogenosis type II, Am. J. Hum. Genet. 56 (1995) 887–897. [6] H. Galjaard, Genetic Metabolic Diseases: Early Diagnosis and Prenatal Analysis, Elsevier/North Holland, Amsterdam, 1980. [7] A.L.E. Montalvo, B. Bembi, M. Donnarumma, M. Filocamo, G. Parenti, M. Rossi, L. Merlini, E. Buratti, P. De Filippi, A. Dardis, M. Stroppiano, G. Ciana, M.G. Pittis, Mutation profile of the GAA gene in 40 Italian patients with late onset glycogen storage disease type II, Hum. Mutat. 27 (2006) 999–1006. [8] M.A. Kroos, R.J. Pomponio, M.L. Hagemans, J.L. Keulemans, M. Phipps, M. DeRiso, R.E. Palmer, M.G. Ausems, N.A. van der Beek, O.P. van Diggelen, D.J. Halley, A.T. van der Ploeg, A.J. Reuser, Broad spectrum of Pompe disease in patients with the same c.-32-13T>G haplotype, Neurology 68 (2007) 110–115. [9] C.R. Scriver, P.J. Waters, Monogenic traits are not simple: lessons learned from phenylketonuria, Trends Genet. 15 (1999) 267–272. [10] K.M. Dipple, E.R.B. McCabe, Modifier genes convert ‘‘simple’’ Mendelian disorders to complex traits, Mol. Genet. Metab. 71 (2000) 43–50. [11] K.M. Dipple, E.R.B. McCabe, Phenotypes of patients with ‘‘simple’’ Mendelian disorders are complex traits: thresholds, modifiers, and systems dynamics, Am. J. Hum. Genet. 66 (2000) 1729–1735. [12] J. Vockley, P. Rinaldo, M.J. Bennett, G.D. Vladutiu, Synergistic heterozygosity: disease resulting from multiple partial defects in one or more metabolic pathways, Mol. Genet. Metab. 71 (2000) 10–18.
L. Gort et al. / Molecular Genetics and Metabolism 92 (2007) 183–187 [13] K.M. Dipple, J.K. Phelan, E.R.B. McCabe, Consequences of complexity within biological networks: robustness and health, or vulnerability and disease, Mol. Genet. Metab. 74 (2001) 45–50. [14] M.M. Hermans, D. van Leenen, M.A. Kroos, C.E. Beesley, A.T. Van Der Ploeg, H. Sakuraba, R. Wevers, W. Kleijer, H. Michelakakis,
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E.P. Kirk, J. Fletcher, N. Bosshard, L. Basel-Vanagaite, G. Besley, A.J. Reuser, Twenty-two novel mutations in the lysosomal alphaglucosidase gene (GAA) underscore the genotype-phenotype correlation in glycogen storage disease type II, Hum. Mutat. 23 (2004) 47–56.
Brief Communication
Glycogen storage disease type II in Spanish patients: High frequency of c.1076-1G>C mutation Laura Gort, M. Josep Coll, Amparo Chaba´s
*
Institut de Bioquı´mica Clı´nica, Hospital Clı´nic, C/ Mejı´a Lequerica s/n, Edifici Helios III, 08028 Barcelona, Spain Centro de Investigacio´n Biome´dica de Enfermedades Raras (CIBERER), Barcelona, Spain Received 9 March 2007; received in revised form 24 May 2007; accepted 24 May 2007 Available online 5 July 2007
Abstract Glycogen storage disease type II is an autosomal recessive disorder of glycogen metabolism due to deficiency of lysosomal acid a-glucosidase. We present the molecular and enzymatic analyses of 22 Spanish GSD II patients. Molecular analyses revealed nine novel mutations. The most common defects were mutations c.-32-13T>G (25%) and c.1076-1G>C (14%) and we report the first homozygous patient for c.1076-1G>C mutation presenting with an infantile form. Alleles bearing mutation c.-32-13T>G are associated with the same haplotype. 2007 Elsevier Inc. All rights reserved. Keywords: Acid a-glucosidase; GAA; Glycogen storage disease; Haplotype analysis; Lysosomal storage disorder; Molecular analysis; Pompe disease
Introduction Glycogen storage disease type II (GSD II, Pompe disease, MIM 232300) is an autosomal recessive disorder of glycogen metabolism due to deficiency of lysosomal acid a-glucosidase (GAA, acid maltase, EC 3.2.1.3). Accumulation of glycogen in the lysosomal apparatus leads to infantile-onset and late-onset (juvenile and adult) forms, but there is a continuum of clinical phenotypes. The infantileonset form is characterized by severe cardiomyopathy, cardiomegaly, profound muscle weakness, delayed motor development and progressive respiratory insufficiency. The juvenile and adult-onset forms present with slowly progressive myopathy and generally without cardiac involvement. A broad spectrum of pathogenic mutations and polymorphic changes has been identified in the lysosomal acid a-glucosidase gene (GAA) (www.pompecenter.nl). Many mutations were identified only once,
although some are common to specific ethnic groups [1– 3]. Mutation c.-32-13T>G is frequent among Caucasian patients presenting late-onset forms [4,5]. Here we present the molecular analysis and enzymatic activity levels of 22 Spanish GSD II patients with identification of 23 mutations, nine of them new. The frequency of the severe mutation c.1076-1G>C is relatively high in both infantile- and late-onset patients. Material and methods Patients Patients were unrelated and came from different regions of Spain. Clinical information was provided by the patient’s physician and diagnosis of GSD II was confirmed by enzymatic determination of GAA activity, except for patient #19 in whom disease was suspected after histological examination of a muscle biopsy and later confirmed by mutation analysis of the GAA gene.
Biochemical assays * Corresponding author. Address: Institut de Bioquı´mica Clı´nica, Hospital Clı´nic, C/ Mejı´a Lequerica s/n, Edifici Helios III, 08028 Barcelona, Spain. Fax: +34 93 227 5668. E-mail address: [email protected] (A. Chaba´s).
1096-7192/$ - see front matter 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.ymgme.2007.05.011
GAA activity was measured in cultured skin fibroblasts and isolated lymphocytes, because of the interfering maltase–glucoamylase activity present in neutrophils, with the fluorogenic substrate 4-methylumbelliferyl-a-D-glucopyranoside [6].
184
L. Gort et al. / Molecular Genetics and Metabolism 92 (2007) 183–187
Molecular analysis
Discussion
Genomic DNA was extracted from whole blood or fibroblasts using standard protocols. Each exon and intron boundaries of the GAA gene was PCR-amplified using self-designed oligonucleotides and standard protocols. PCR products were directly sequenced. Gene nucleotide numbering is according to sequence RefSeq NM_000152, with +1 as A of the ATG start codon. The ATG codon represents +1 for the amino acid numbering according to a-glucosidase preprotein sequence NP_000143.
The most frequent mutation in Spanish patients is the splicing mutation c.-32-13T>G (total allele frequency 25% and 34.4% of late-onset alleles) which has been reported to be the most common among the Caucasian late-onset affected population [4]. This frequency is low when compared to previous reports: 68% [4] and 42.3% [7]. In our study all the patients carrying this mutation also presented with the juvenile or adult form of the disease, corroborating the suggestion that this mutation is a mild one and that clinical manifestations depend upon the severity of the mutation in the other allele [7]. Within a clinical phenotype, patients bearing this mutation, even when associated with the same haplotype, may manifest initial symptoms at different ages and may present different disease severity, indicating that secondary factors may influence the clinical course [8–13]. The second most frequent mutation in the Spanish GSDII population is the splicing change c.1076-1G>C, with allele frequency of 14%; this mutation has not been previously detected in larger series of patients except at heterozygosity in one patient and it is presumed to be causative of an insertion (r.1076-79_1195+89ins) [7]. In the Spanish series mutation c.1076-1G>C was found at homozygosis in an infantile patient, with severe cardiomyopathy, cardiomegaly and hepatomegaly. This would indicate that mutation c.1076-1G>C is severe, and the fact that patients carrying this mutation present with the juvenile or adult form of the disease may be due to the mildness of the mutation on the other allele. Other novel mutations have been found at heterozygosity only in one or two patients so that expression studies are necessary to establish the severity of these changes. Among previously reported mutations, mutation c.546G>C is interesting. Although it does not change the amino acid in position 182 (Thr), it affects the last nucleotide of exon 2 and it is presumed to cause leaky splicing [14]. All 11 alleles bearing mutation c.-32-13T>G in Spanish patients are associated with the same haplotype, the DHRGEVVT haplotype as named after Kroos et al. [8], and we have extended this haplotype with 12 more informative polymorphic markers. Although this mutation has occurred more than once on different haplotypes, the presence of a single haplotype associated with this change in Spain suggest a possible founder effect. Mutation c.10761G>C was carried by six alleles (five patients) but only in one case this mutation was associated with the same haplotype as mutation c.-32-13T>G, while in five alleles mutation is associated with a different haplotype. This last haplotype is based on the first most common GAA core haplotype (DRHGEIVT according to [8]), except for position E (c.2065G) that in our five alleles is K (c.2065A). These results would suggest that this frequent mutation among Spanish patients occurred at least twice although most patients originate from the same region.
Results Biochemical findings Some correlation was found between fibroblast residual GAA activity and clinical phenotype. Mean ± SD GAA activity levels were 2.0 ± 0.9%, 7.1 ± 5.4% and 20.2 ± 7.7% of mean control values in the infantile, juvenile and adult patients, respectively, although the level of residual activity ranged widely even in patients with the same disease phenotype. In contrast, in lymphocytes, residual activity ranged from 1.9% (juvenile patient #24) to normal values (adult patient #34). GAA activity, genotypes and phenotypes of the 22 patients are summarized in Table 1. Mutation analysis We analysed 22 unrelated GSDII patients to determine the molecular defect in the GAA gene. We found nine novel and fourteen already described mutations in these patients. The novel mutations are p.Q115X (c.343C>T), p.L169P (c.506T>C), p.L398fsX107 (c.1192dupC), p.Y455C (c.1364A>C), p.P482L (c.1445C>T), p.A644P (c.1930G> C), p.D645Y (c.1933G>T), p.I752del (c.2255_2257delTCA) and p.E867fsX19 (c.2600_2604delTGCTGinsA). None of the missense mutations were found in 100 control chromosomes. The most common defects were c.-3213T>G and c.1076-1G>C mutations accounting for 25% and 14% of total alleles, respectively. Polymorphisms and haplotype analysis Six novel nondeleterious changes in the GAA gene were detected: c.693-49C>T (intron 3); c.852G>A (exon 4); c.1075+13C>T (intron 6); c.2331+151C>T (intron 16) and c.2331+293A>T (intron 16). We also studied associations between mutations c.-32-13T>G and c.1076-1G>C and specific haplotypes (Table 2). All eleven alleles with mutation c.-32-13T>G are presumably associated with the same haplotype. In contrast, for c.1076-1G>C mutation, identified in six alleles, the change was associated with one haplotype in five alleles while the other allele was associated with the same haplotype as mutation c.-32-13T>G. Haplotype association of each mutation was deduced when polymorphic markers revealed homozygosity, and in cases of heterozygosity consistency was checked.
Table 1 Genotype, phenotype and GAA activity in 22 GSD II patients Allele 1
Allele 2
Phenotype
Age at onset
Main clinical traits
Residual GAA activity (%)
2 4 6 7 11 50 5 9 21 24 3 19 22 1 8 12 15 34 47 54
p.L169P c.1076-1G>C c.2481+102_2646+31del p.N470del p.L552P p.Y455C c.-32-13T>G c.-32-13T>G c.-32-13T>G c.-32-13T>G c.1076-1G>C c.1076-1G>C p.P482L c.-32-13T>G c.-32-13T>G c.-32-13T>G c.-32-13T>G c.-32-13T>G c.-32-13T>G c.-32-13T>G
p.D489N c.1076-1G>C c.2646+2T>A p.M408V c.2600_2604delinsA p.G638W p.L552P c.2255_2257delTCA p.G638W p.G309R p.R725W p.R725W p.Q115X p.Y292C p.D645Y c.1192dupC p.R725W p.G478R c.2600_2604delinsA c.546G>C
Infantile Infantile Infantile Infantile Infantile Infantile Juvenile Juvenile Juvenile Juvenile Juvenile Juvenile Juvenile Adult Adult Adult Adult Adult Adult Adult
Birth 4m 4m Birth 6m 2m 6y 3y 5y 2y6m 3y 3y 2y (60y) 30y 31y (44y) NA 35y (65y)
3.0 (F) 3.5 (F) 0.6 (F) 1.8 (F) 2.3 (F) 10 (L);2.4 (F) 4.6 (L) ; 11 (F) 11.4 (F) 9.0 (F) 1.9 (L) 1.1 (F) NA 35 (L) 17 (F) 20 (F) 28 (F) 5.2 (F) 90 (L) 13 (L); 13 (F) 15 (L); 21 (F)
14 17
c.1076-1G>C c.1076-1G>C
p.Q115X p.A644P
Adult Adult
(34y) (34y)
HM, MC H, HM, MD, MC H, HM, MC, leukopenia H, HM, cardiomegaly NA NA H,MD, skeletal abnormalities NA Muscular atrophy, torax deformity H, muscular weakness HM, MD, splenomegaly H, respiratory failure H, MC MD, MC Respiratory muscle weakness Myopathy, muscle weakness MD, vacuolar myopathy NA Strabismus, progressive muscular weakness Chronic pneumonopathy, ischemic cardiopathy, basal dyspnea Muscle weakness, respiratory insufficiency, difficulty feeding H, glaucoma, Marfan syndrome
13 (L) 5 (L)
L. Gort et al. / Molecular Genetics and Metabolism 92 (2007) 183–187
Patient
Gene nucleotide numbering is according to sequence RefSeq NM_000152, with +1 as A of the ATG start codon. Age at diagnosis, in parentheses. NA, not available; H, hypotonia; HM, hepatomegaly; MD, muscular dystrophy; MC, myocardiopathy; F, fibroblasts; L, lymphocytes.
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References
T T C
C C G
A A G
G G A
Ins
T T C
G G A
G G A
A A G
G G C
C C A
G G A
A A G
G G A
T T C
G G A
Ins A A G
The authors thank all the physicians who referred the patients, Drs.: Azan˜a, Barreiro, Castro, Civantos, Colomer, Blanco, Dı´az, Ferna´ndez Villalba, Gala´n, Garcı´a Silva, Gayoso, I. Gonza´lez, N. Gonza´lez, Hermida, Illa, Lo´pez Coronas, Lo´pez de Munain, Lo´pez Va´zquez, Lorente, Martı´nez Garcı´a, Morera, Olive´ Planas, Ortola Devesa, Pascual Pascual, Pe´rez-Sheriff, Rojas, Tamariz-Martel, as well as to A.M. Ban˜on Herna´ndez (Asociacio´n Espan˜ola de Enfermos de Glucogenosis). We thank J. Duque, J. Jarque, H. Selle´s and A. Valle for excellent technical assistance. This study was supported in part by FIS (Redes tema´ticas, G03/054 REDEMETH and 05/1182).
G G A
Acknowledgments
c.-32-13T>G 11 c.1076-1G>C 1 5
c.596 c.668 c.858 c.858 A/G G/A +7ins7 +30T/C Number c.324 c.547-4 of alleles T/C C/G
Table 2 The Spanish most frequent mutations associated haplotypes
c.955 c.1203 c.1327- c.1438- c.1551 c.1888 c.2040 c.2065 c.2133 c.2331 c.2331 c.2338 c.2998insG +12G/A A/G 18A/G 19G/C +49C/A +21G/A +20A/G G/A A/G +20G/A +24T/C G/A
186
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