The proportion of patients with PD from the ones on suspection of PD in other similar studies in caucasic population was 0.29%  and 2.2%  In our population, we tested 2637 samples with suspection and a total of 17 new patients of PD were found (0.64%).
The median age at diagnosis in this set of patients was 38 years, this is in line with other authors who maintain that clinical manifestations in LOPD may present from the first decade to the seventh decade of life and the median age at diagnosis is 38 years [10, 11] In our population, three patients were asymptomatic [patients #2, #3 and #6]. All of them were included in the study because they had a family member with PD and they probably show no clinical symptoms because they are still too young. Patient #2 and patient #3 are sisters and they are seven and nine years old respectively and patient #6 is a nineteen year old female. The early diagnosis of LOPD in asymptomatic subjects is rare and it may be explained by the delay in the first clinical manifestations as described in previous studies 11 which shows the importance of family studies for preventive follow-up [12, 13]
The clinical symptoms in our cohort were similar to the classical findings in Pompe disease studies [14, 15]. We confirm that the most common symptom in LOPD is muscle weakness and it was present in all patients except the asymptomatic ones because they are early diagnosis prescribed for family study. Elevated CPK levels and respiratory distress were the next most frequent symptoms. Myalgia, dysphagia or hypotonia were less frequent symptoms in our population (12,5%). Patient #1 [two days of age], showed an hypertrophic cardiomyopathy, the most frequent manifestation in IOPD as reported the literature  PD presents a great clinical heterogeneity, even in patients with the same mutation. Therefore, the type and degree of manifestations of each individual could depend on the residual enzymatic activity and its interaction with other genetic or epigenetic factors.
In accordance with others studies, our results confirm that the mutations are distributed throughout the entire gene. [17, 18]. As published in the bibliography, the gene has three critical regions: exon 2, which includes start codon, exon 10 and 11 where the evolutionarily conserved catalytic site domain is contained, and exon 14 which includes a highly conserved region. Two variants of our study were detected in exon 2, none in exons 10, 11 or 14. The rest of the variants are distributed by almost all exons as shown in the table 2. Due to the NGS boom, it is expected that more variants of uncertain significance will be explained in the future.
Sequence analysis of the complete coding region of the GAA gene revealed 14 different variants from 17 patients including nine missense mutations (26.4%), one nonsense mutation (2.9%), three deletion or insertion (17.6%), eighteen splicing variants (52.9%).
Similar to others studies, the splice-site mutation c.-32-13T > G was the most frequent mutation found in our cohort. As is published in the literature, the intronic variant is the most common in Caucasian populations and it is present in 40–70% of the alleles in patients affected with PD . In this study, it was seen in all patients except patient #1 and patient #5 (17 alleles, 50%). Patient #13 and patient #16 presented the variant in homozygosis. This variant is located in the 3´splice region and it causes aberrant splicing of the GAA gene. For this reason, the splicing variant c.-32-13T > G is considered pathogenic [19–21]
The next most frequent mutation present in our population was c.236_246delCCACACAGTGC. It was described by Palmer  in a patient who presented a severe infantile-onset Pompe disease. In concordance with the previous study, we encountered the delection in homozygosis in patient #1. Patient #1 had a sister with diagnosed PD who died at nine months of age and for whom we do not have the results of the genetic study, the clinical information refers to parents as carriers of the disease. It was found too in heterozygosis in patient #10, a forty-six years old man. The presence of the homozygous variant could be established as providing a more serious effect or being indicative of a worse prognosis.
The mutation c.1396_1397insG was identified in two heterozygotes patients, they were asymptomatic sisters (patient #2 and #3) who showed the same genotype (c.-32-13T > G + c.1396_1397insG) very young to present the PD clinical symptoms (7 and 9 years old respectively). The variant is described as cause of PD creating a frame shift starting at codon Val466 and a stop codon in 39 position downstream . The rest of the variants (c.281_282delCT; c.655G > A; c.875A > G; c.925G > A; c.1655T > C; c.2104C > T; c.2237G > C) had already been described in the literature as pathogenic were shown only once and, therefore, were less frequent in our population.
This study contributed to the identification of four new probably pathogenic variants which had not been described previously in the literature (c.1328A > T; c.1831G > A; c.2819C > A; c.1889-1G > A).
The substitution c.1328A > T (p.Asp443Val) was detected in exon 9 of patient #5. The missense variant produces a change in the protein and replaces aspartic acid with valine at codon 443. There are physicochemical differences between these amino acids. Acid aspartic is neutral and polar and valine is neutral and non-polar. This could modify the conformation of the protein and affect its function. Other missense variants have been reported as pathogenic in nearby codons [24, 25]. This findings suggests that this variant contributes to disease.
The missense variant c.1831G > A is located in exon 13 and it was shown in heterozygosis in patient #4. This substitution (p.Gly611Ser) replaces glycine with serine at codon 611. Glycine is non-polar and serine is polar. These physicochemical differences can alter the structure of the protein and could affect its function. On the other hand, it is the second variant discovered in this codon. The mutation c.1832G > A (p.Gly611Asp), which also change glycine for a polar amino acids, was described in previous study and was reported as pathogenic variant .
We detected the nonsense variant c.2819C > A in patient #9. This variant generates a slightly truncated protein (p.Ser940Ter). It was assumed to be deleterious since the stop codons of other proteins were detected upstream of this and were known to result in a complete loss of enzyme activity. The variant c.2741delinsCAG [p.Gln944*fs30] produces a premature stop codon in aminoacid 944, was previously described by van Gelder . in patients that did not present any activity of α-glucosidase. This leads us to think that a previous stop codon will also generate damage to the protein.
The splicing variant c.1889-1G > A in the intron 13 was detected in patient #11. As Anna  published, in general, mutations in the canonical acceptor and donor sites affect strongly conserved sequences that define exon-intron boundaries. Therefore, any variants in these canonical sequences might alter interaction between premRNA and proteins involved in the intron removal.