HUMAN MUTATION Mutation in Brief #1007, 29:E27-E36, 2008 (Online)
MUTATION IN BRIEF
© 2008 WILEY-LISS, INC.
Received 29 October 2007; accepted revised manuscript 14 January 2008.
Molecular and Functional Characterization of Eight
Novel GAA Mutations in Italian Infants with Pompe
MG Pittis1, M Donnarumma2#, ALE Montalvo1#, S Dominissini1, M Kroos3, C Rosano4, M Stroppiano2,
MG Bianco2, MA Donati5, G Parenti6, A D’Amico7, G Ciana1, M Di Rocco8, A Reuser3, B Bembi1, and
#These authors contributed equally to the work
1Unità di Malattie Metaboliche, IRCCS Burlo Garofolo, Trieste; 2Laboratorio Diagnosi Pre-Postnatale Malattie
Metaboliche and 8U.O. Pediatria II, IRCCS G.Gaslini, Genova, Italy; 3Department of Clinical Genetics, Erasmus
MC, Rotterdam, The Netherlands; 4Bioinformatics and Structural Proteomics - National Institute for Cancer
Research (IST), Genova, Italy; 5Dept of Paediatric Neurology, Meyer Hospital, Florence, Italy; 6Department of
Pediatrics, Federico II University, Naples, Italy; 7Unita' di Medicina Molecolare, Dipartimento Laboratori, IRCCS
Bambino Gesù, Roma, Italy
*Correspondence to Dr Mirella Filocamo, Lab Diagnosi Pre-Postnatale Malattie Metaboliche, Istituto G. Gaslini,
Largo G. Gaslini 5 (16147) Genova, Italy. Tel: +39 010 5636792; Fax: +39 010 383983;
Communicated by William S. Sly
We characterized 29 unrelated patients presenting with the severe form of Pompe disease
(Glycogen Storage Disease Type II, acid maltase deficiency) and identified 26 pathogenic
mutations divided over 28 different genotypes. Among the eight new mutations, five were
exonic point mutations (c.572A>G, c.1124G>T, c.1202A>G, c.1564C>G and c.1796C>A)
leading to codon changes (p.Y191C, p.R375L, p.Q401R, p.P522A and p.S599Y); two were
intronic point mutations (c.-32-3C>A and c.1636+5G>C) affecting mRNA processing; one
was a single base deletion (c.742delC) generating a truncated protein (p.L248PfsX20). A
comprehensive evaluation, based on different methodological approaches, confirmed the
detrimental effect of the eight mutations on the protein and its function. Structural
alterations potentially induced by the five missense mutations were also predicted through
visual inspection of the atomic model of the GAA protein, in terms of both function and
spatial orientation of specific residues as well as disturbance generated by amino acid
substitutions. Although the remarkable heterogeneity of the mutational spectrum in Pompe
disease was already known, our data demonstrate and confirm the power of molecular and
functional analysis in predicting the natural course of Pompe disease. © 2008 Wiley-Liss, Inc.
KEY WORDS: Pompe disease; Glycogen Storage Disease type II; infantile onset; GAA mutational spectrum; α-
Pompe disease (Glycogen Storage Disease Type II, acid maltase deficiency, (MIM# 232300)) is an autosomal
recessive progressive muscular disorder caused by the deficit of acid α-glucosidase (GAA; E.C.126.96.36.199) that
results in impaired lysosomal degradation and accumulation of glycogen. Infantile Pompe disease manifests soon
E28 Pittis et al.
after birth and the most frequent symptoms include severe muscle weakness, generalized hypotonia, failure to
thrive, cardiomegaly/cardiomyopathy and respiratory insufficiency, leading to death within the first year of life
[Hirschhorn & Reuser, 2001; Raben et al., 2002; van den Hout et al., 2003; Kishnani & Howell 2004; Kishnani et
al., 2006]. Some infantile patients have less severe cardiac involvement without cardiac-output obstruction, survive
longer and die because of pulmonary infections with secondary respiratory insufficiency [Slonim et al., 2000;
Winkel et al., 2005; Kishnani et al., 2006].
Recently, enzyme replacement therapy (ERT) has been registered for Pompe disease treatment. The first results
showed that recombinant human α-glucosidase (Myozyme®) may effectively slow or reverse the course of the
disease [Kishnani et al., 2007]. The best therapeutic results are achieved when ERT is started early in the course of
symptom development and before irreversible muscular damage has occurred. Increasing natural history data on
infantile Pompe disease is needed to provide an accurate patient management and to determine the indication and
timing of treatment. Other approaches, still in a pre-clinical stage, include chaperone-mediated therapy aimed at
enhancing the residual activity and improving the transport of mutant GAA species [Okumiya et al., 2007; Parenti
et al., 2007].
The GAA gene (MIM# 606800) is mapped to human chromosome 17q25.2-25.3; the enzyme, synthesized as an
inactive precursor of 110 kD, is transported to the pre-lysosomal and lysosomal compartment via the mannose 6-
phosphate receptor where it is processed into the 95 kD intermediate and the fully active forms of 76 and 70 kD
[Hoefsloot et al., 1990a,b; Martiniuk et al., 1991; Hirschhorn & Reuser, 2001; Moreland et al., 2005]. A large
number of mutations in GAA gene have been described up to date [http://www2.eur.nl/fgg/ch1/pompe]. Some
years ago we started to make an inventory of the GAA sequence variations in the Italian population and reported
the genotypes of eight infants [Stroppiano et al., 2001; Pittis et al., 2003; Montalvo et al., 2004] and a larger series
of older patients with Pompe disease [Montalvo et al., 2006]. Here, we completed the mutational analysis in 29
unrelated infants, in which we identified and functionally characterized eight novel mutations among which there
were five missense mutations, one small deletion and two intronic sequence variations affecting mRNA splicing.
The effect of another mutation, potentially interfering with mRNA splicing, is discussed. This study, integrated
with the previous ones, provides a complete picture of the molecular basis of Pompe disease in Italy and represents
a contribution to the understanding of clinical diversity, natural course and efficacy of enzyme replacement and
other modes of therapy.
MATERIALS AND METHODS
We studied 29 unrelated patients affected by Pompe disease coming from different parts of Italy (16 females,
11 males, and 2 fetuses). Twenty-seven patients presented with a fatal clinical course of the disease, including
myocardiopathy and severe hypotonia, while two patients have survived receiving enzyme replacement therapy.
The diagnosis was based on clinical data and confirmed by reduced lysosomal α-glucosidase activity in
different tissues (fibroblasts and/or blood samples). The age at diagnosis varied from 2 to 36 months. Two cases
were diagnosed prenatally in two unrelated families, in which the index cases were deceased without a laboratory
confirmation of the clinical diagnosis.
Enzyme activity assay
α-Glucosidase activity was measured using the fluorogenic substrate 4-methylumbelliferyl-α-D
glucopyranoside (Sigma, St. Louis, MO, USA) [Hermans et al., 1991]. Protein concentration of the samples was
determined by the Lowry method. Enzymatic activity was expressed as nanomoles of substrate hydrolyzed per
milligram of total protein per hour. All assays were done in triplicate from at least three separate transfections.
GAA mutation analysis
Genomic DNA was isolated from cultured fibroblasts or peripheral blood leukocytes using standard protocols or
suitable kits, QIAmp DNA blood mini kit (Qiagen GmbH, Hilden, Germany), or Nucleon BACC3 kit for blood
and cell cultures (Amersham Biosciences Inc., Freiburg, Germany). Coding GAA exons and their flanking regions
were PCR amplified as described elsewhere [Ko et al., 1999] and sequenced with the ABI PRISM Big Dye
Terminator Cycle Sequencing Kit (Applied Biosystems, Warrington, UK) following the manufacturer’s
Infantile Pompe Disease in Italy E29
instructions. Mutations were confirmed by sequencing duplicate PCR products and by the DNA analysis from
parents and relatives.
Total RNA was isolated from cultured fibroblasts using Trizol Reagent (Gibco, Paisley, UK) according to the
manufacturer's instructions. For RT-PCR analysis the first strand cDNA was synthesized using random hexamer
primers and subsequent amplification was done in six overlapping fragments as described by Hermans et al.
[Hermans et al., 1997]. To analyze the c.-32-3C>A mutation, the 5’GTTGTTCAGCGAGGGAGGCTCT3’ was
used as forward primer and the 5'AAGGGTGAGACCCGTAGAGGTTCG3' as reverse primer. To analyze
c.1636+5G>C mutation the 5’ATCCTGCCATCAGCAGCTCG3’ was used as forward primer and the
5’GGAGATCACAAATGGGCGTG3’ as reverse primer.
Site directed mutagenesis
Site-directed mutagenesis was carried out using the Quickchange Site-Directed Mutagenesis Kit (Stratagene,
Cedar Creek, TX, USA) according to the manufacturer's instructions. Each clone was entirely sequenced to
confirm that no other mutations were introduced by the PCR-based mutagenesis procedure.
Cell culture and transient transfection
Patient fibroblasts obtained from skin biopsies were cultured in RPMI 1640 supplemented with 10% fetal calf
serum, 2mM L-glutamine and 50 mg/ml penicillin/streptomycin (Gibco, Paisley, UK).
For in vitro expression assays the Ad5-SV40 immortalized human GAA-deficient fibroblast cell line (kindly
provided by Dr. Martiniuk) was grown in monolayers in RPMI 1640 medium supplemented with 10% fetal calf
serum and 50 mg/ml penicillin/streptomycin (Gibco, Paisley, UK). Alternatively, COS-7 cells were used for this
purpose, as previously described [Hermans et al., 2004]. The GAA deficient cells were transfected with wild type
and mutant constructs with a standard calcium/phosphate procedure using 4 μg of total plasmid DNA Endofree
purified (Sigma, St. Louis, MO, USA) following the manufacturer’s instructions. Cells were harvested after 48 h
and assayed for GAA activity and synthesis (by Western blotting).
Western immunoblot analysis
Mutations p.Y191C, p.R375L, p.Q401R, p.P522A, p.S599Y. Cell lysates (15 μg of protein/lane) were resolved
on 10% SDS-PAGE gels, transferred onto a nitrocellulose membrane (Biorad, Hercules, CA, USA). Blotted
membranes were probed with an antiserum against α−glucosidase as described elsewhere [Montalvo et al., 2004].
An anti-rabbit HRP conjugated antibody (DAKO, Glostrup, Denmark) was used as a second antibody and
developing was performed by enhanced chemoluminescence (ECL Amersham Biosciences, Buckinghamshire,
UK). The antibody recognized all the forms of α−glucosidase: the precursor of 110 kD, a processing intermediate
of 95 kD and the mature forms of 76 and 70 kD.
The three dimensional models of the wild type protein and the enzyme with amino acid mutations were built
with the Swiss-model server (1-3) using, as template, the coordinates of the Sulfolobus solfataricus α-glucosidase
catalytic domain (PDB 2G3M) [Ernst et al., 2006]. All the models were energy minimized using the program
Discover3 from the InsightII program suite (Accelrys, Inc., San Diego, CA, USA) and stereochemistry optimized
by the program REFMAC5 (Murshudov et al., 1997). Graphical representation and amino acid substitutions were
carried out using the program O [Jones et al., 1991].
All mutations are described according to mutation nomenclature, considering nucleotide +1 the A of the first
ATG translation initiation codon [den Dunnen and Antonarakis, 2000; den Dunnen and Paalman, 2003]
(www.hgvs.org/mutnomen). Nucleotide numbers are derived from cDNA GAA sequence (EMBL/GenBank/
DDBJ; accession number Y00839.1and M34424).
E30 Pittis et al.
RESULTS AND DISCUSSION
We performed the complete molecular analysis of the GAA gene in 29 unrelated Italian patients with infantile
Pompe disease. Table 1 summarizes the 28 distinct genotypes encountered in our series, in which the majority of
the patients (66%) were compound heterozygous and only ten patients were homozygous.
Table 1: Genotypes Encountered in the 29 Italian Pompe Patients
1 F 5 mos c.525delT (p.E176fsX45)
2 F 2 mos c.525delT (p.E176fsX45)
3 F NA c.525delT (p.E176fsX45)
4 F 6 mos c.525delT (p.E176fsX45)
5 M 5 mos c.525delT (p.E176fsX45)
6 M 12 mos c.525delT (p.E176fsX45)
7 F 10 mos c.2481+102_2646+31del (p.G828_N882del)
8 F 3 mos c.2481+102_2646+31del (p.G828_N882del) c.1202A>G (p.Q401R)
Pt no= patient number; F=female; M=male; mos=months; NA=not available; *EMBL/GenBank/ DDBJ:accession number
Y00839.1 and M34424; #dead at 8 months; § This case was diagnosed at the age of 3; she had been described as always
presenting difficulties to run and jump. At diagnosis she showed failure to thrive, facial weakness, rhinolalia, limb girdle
muscular weakness, walking, climbing and jumping difficulties. Hypertrophic cardiomyopathy had been detected. At the
age of 6, after two years of enzyme replacement therapy, the patient is still alive with stable muscle weakness and
Overall, the mutational profile was characterized by 26 different mutant alleles including eight previously
unreported, that are: c.-32-3C>A, c.572A>G, c.742delC, c.1124G>T, c.1202A>G, c.1564C>G, c.1636+5G>C,
c.1796C>A. One allele has been partially characterized and three others remain still unknown.
Table 2 reports location, characteristics and allele frequency of all mutations. The most prevalent mutation was
c.525delT accounting for 13.8% of the alleles. The other mutations with a relatively high frequency were p.L552P
(8.6%), p.E262K, p.L355P, p.G643R and p.G828_N882del (6.8%, each). All of these mutations were previously
c.784 G>A (p.E262K)
Infantile Pompe Disease in Italy E31
reported and our findings confirm their panethnic nature. Of considerable interest is the occurrence of p.L552P as
the second most frequent mutation. In fact, “in vitro” studies, carried out on patients’ fibroblasts have recently
demonstrated a significant increase of GAA activity as well as an improved trafficking of the mutant enzyme to
lysosomes after treatment with imino sugar chaperones [Parenti et al., 2007].
The new mutations comprise 20.3% of the alleles, which is indicative for the genetic heterogeneity in Pompe
disease. Apart from c.1124G>T (3 times) and c.1564C>G (2 times), the remaining new alleles (c.1202A>G,
c.1636+5G>C, c.1796C>A, c.-32-3C>A and c.572A>G) were encountered only once.
Among the new mutations, the five exonic point mutations c.572A>G, c.1124G>T, c.1202A>G, c.1564C>G
and c.1796C>A lead to codon changes (p.Y191C, p.R375L, p.Q401R, p.P522A and p.S599Y, respectively); the
two intronic point mutations c.-32-3C>A, and c.1636+5G>C affect mRNA processing; the single base deletion
c.742delC causes frame shift and generates a premature stop codon and a truncated protein (p.L248PfsX20).
The functional relevance of the two intronic mutations (c.-32-3C>A, and c.1636+5G>C) was determined by
carrying out reverse transcriptase-polymerase chain reaction (RT-PCR) analysis on mRNAs from the cultured
fibroblasts of the respective patients (#12 and 28).
In patient 12 with genotype [c.-32-3C>A]+[c.1655T>C], the RT-PCR analysis showed that the intronic
mutation caused the loss of 579 bp (Fig. 1A). The anomalous transcript was predicted, therefore, to translate into a
Table 2: Mutation Profile of the GAA Gene in the Italian Pompe Patients
Location cDNA mutation*
Exon 5 c.877G>A
Exon 6 c.1064T>C
Intron 9 c.1437+2T>C
Exon 10 c.1465G>A
Exon 12 c.1655T>C
Exon 16 c.2237G>A
Intron 16 c.2331+2T>C
Exon 17 c.2432delT
Intron 17-18 c.2481+102_2646+31del
Hermans, et al., 2004
Hermans, et al., 1994
Fernandez-Hojas, et al., 2002
Pittis, et al., 2003
Fernandez-Hojas, et al., 2002
Hermans, et al., 2004
Hermans, et al., 2004
Stroppiano, et al., 2001
Montalvo, et al., 2006
Bodamer, et al., 2002
Vorgerd, et al., 1998
Hermans, et al., 1993
Huie, et al., 1998
Beesley, et al., 1998
Hermans, et al., 1997
Amartino, et al., 2006
Huie, et al., 1994a
* cDNA reference sequence: EMBL/GenBank/ DDBJ,accession number Y00839.1 and M34424. For cDNA numbering +1
corresponds to the A of the first ATG translation initiation codon; red and bold type marks new mutation
E32 Pittis et al.
shorter peptide lacking the first 182 amino acids (p.M1_T182) comprising the 27 residues of signal sequence, the
42 residues of the propeptide and the first 113 amino acids of the mature protein (from 70 to 182).
In patient 28, the c.1636+5G>C mutation in the splice donor site of intron 11 caused the incorporation of the
complete intron 11 in the GAA messenger (r.[1636+5g>c;1636_1637ins1636+1_1636+957]) (Fig. 1B). As a
consequence, this splice site mutation is predicted to insert 146 non-canonical codons including a premature stop
codon at downstream position (p.G546_V547ins145X146).
Figure 1. RT-PCR analysis of GAA mRNA
Patient 12 (Fig. 1A). As a consequence of the
mutation c.-32-3C>A, the analysis revealed an
anomalous transcript fragment of 427bp besides
the expected one of 1006bp.
Patient 28 (Fig. 1B). In this case the mutation
c.1636+5G>C resulted in an anomalous transcript
fragment of 1427bp besides the expected one of
Ma=marker, 1kb Plus DNA Ladder; Mb=marker,
φX 174 DNA HaeIII-digested; lane 1=patient
sample; lane 2=control sample.
Finally, another mutation possibly altering RNA expression was hypothesized on the basis of RT-PCR analysis.
By sequencing the genomic DNA, patient 26 was found to be heterozygote for c.572A>G, but a second mutation
was not detected. Further investigations were therefore carried out by RT-PCR analysis. Sequencing of RT-PCR
products from three different sets of primers, clearly showed only the transcript with the mutation c.572A>G, thus
suggesting that a still undiscovered genomic lesion is responsible for the lack of the corresponding mutant
The five novel single base substitutions (c.572A>G, c.1124G>T, c.1202A>G, c.1564C>G and c.1796C>A)
seem also functionally important. All changes are nonconservative (p.Y191C, p.R375L, p.Q401R, p.P522A,
p.S599Y) with substitutions resulting in chemical-physical property changes including charge, mass and side chain
However, to address the question of the
potential impact of these missense mutations on
protein function we performed in vitro expression
experiments. The mutant constructs p.Y191C,
p.R375L, p.P522A and p.S599Y as well as wild
type GAA cDNA were transiently transfected in the
Ad5-SV40 immortalized human GAA-deficient
fibroblast cell line. Alternatively, COS-7 cells were
used for mutant p.Q401R and wild type GAA
cDNAs. All mutants tested expressed from zero to
extremely low residual GAA activity (Table 3).
These findings were in agreement with Western blot analysis (Fig. 2). As shown in Figure 2A, p.S599Y was
retained as the inactive 110-kD precursor while no immune reactive protein could be detected for p.Y191C,
p.R375L and p.P522A mutant constructs. Similarly, western blot analysis of mutant p.Q401R GAA cDNA
expressed in COS cells demonstrated that there was hardly any GAA protein detectable in the cells transfected
with this mutant construct (Fig.2B).
Table 3: Enzymatic activity of GAA missense mutations
*The activity of mutant GAA species are expressed as % of
normal after subtraction of the activity of the respective mock-
4MU% of wild-type*
Infantile Pompe Disease in Italy E33
Figure 2. Western blot analysis of wild-type (WT) and mutant species in transfected cells. Ad5-SV40
immortalized human GAA-deficient fibroblast cell line was transfected with the mutant (p.Y191C, p.R375L, p.P522A,
p.S599Y) GAA cDNA constructs at Burlo Garofolo Institute (Fig. 2A). COS-7 cells were transfected with the mutant
(p.Q401R) GAA cDNA construct at Erasmus Institute (Fig. 2B). All the forms of GAA can be detected in WT, while no GAA is
detected in the mock-transfected negative control (M). The mutation p.S599Y was retained as the inactive precursor of 110-kD
while no immune reactive protein could be detected for p.Y191C, p.R375L, and p.P522A mutant constructs (Fig. 2A). A very
faint band of 110 kD was observed inside the p.Q401R cells but not in the respective culture medium (Fig. 2B).
Up to date the crystal structure of human GΑΑ has not yet been solved. To predict structural alterations
potentially induced by these missense mutations, we built atomic models of human wild-type α-glucosidase and
the enzyme with substituted residues by homology modeling, as previously reported [Tajima et al., 2007]. The
amino acid replacements were predicted through visual inspection of this atomic model, in terms of both function
and spatial orientation of specific residues as well as disturbance generated by amino acid substitutions (Fig. 3).
Particularly, p.R375L mutation occurred on the protein surface, in a zone shaped as a narrow well. The
Cells CellsMedium Medium
replacement of the Arginine, a polar and charged
residue by a hydrophobic residue, such as Leucine,
might alter this region by closing the well to prevent
water access to the protein core; additionally the
residue change would lead to the loss of a positive
charge on the protein surface. Mutation p.Q401R,
on the contrary, would add a positive charge by
replacing a polar (but not charged) residue.
Additionally, the new Arginine (401R) being in
close contact with R436 could disrupt the formation
of a probable disulphide bond between C647 and
C658 (residues conserved among mammalian alpha-
glucosidases) thus altering the correct fold of the
protein [Tajima et al., 2007]. P522 is well
conserved too and it is located at the end of a β-
strand, before an α-helical region of the protein.
Proline confers a local rigidity to the protein
backbone and its substitution with an Alanine (p.P522A) could dislocate the secondary structure of the region.
Mutation p.S599Y occurs on the protein surface. Although both residues are polar and the bigger side chain of the
tyrosine could easily relocate in the solvent, the presence of F649 and F603 in this area could easily bring to the
formation of p-p stacking interactions between the aromatic rings of these residues. Finally, using our model it was
not possible to directly inspect the mutation p.Y191C. This mutation, however, might interfere with the correct
folding of the protein because of the replacement of a tyrosine (polar residue having a large side chain) by a
reactive cysteine having a small side chain.
Although in empirical manner, the three-dimensional structure predicted incorrect protein folding for all
missense mutations. Recent studies have shown that the endoplasmic reticulum (ER)-associated degradation
system (ERAD) is operative in degrading mis-folded lysosomal proteins and contributes that way to the degree of
Figure 3: Ribbon representation of a human acid α-glucosidase
model structure. Mutation sites are reported in yellow
E34 Pittis et al.
lysososmal enzyme deficiency [Ron and Horowitz, 2005]. Hence, a similar situation may occur in Pompe disease.
It would explain why mutant forms of GAA with a single missense mutation are not detected by Western blotting.
To conclude, we characterized 29 unrelated patients presenting with the severe form of Pompe disease and
identified 26 pathogenic mutations divided over 28 different genotypes. All seventeen previously reported
mutations were known to completely abolish the GAA activity. A comprehensive evaluation of the molecular and
functional characteristics of the eight novel mutations revealed that they too were pathogenic and in various
combinations associated with infantile Pompe disease. Although the remarkable heterogeneity of the mutational
spectrum was already known, our data demonstrate and confirm the power of molecular and functional analysis in
predicting the natural course of Pompe disease.
The samples were obtained from the “Cell Line and DNA Bank from Patients affected by Genetic Diseases”
collection (http://www.gaslini.org/labdppm.htm) supported by Italian Telethon grants.
This work was supported by a grant from Italian Health Ministry (Italia-USA RF526 D/47).
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