Genotype-Phenotype correlation of 17 cases of Pompe Disease in Spanish Patients and Identication of 4 Novel Mutations

Background: Pompe disease (PD) is an autosomal recessive metabolic disorder caused by mutations in the acid α-glucosidase gene (GAA) that produces defects in the lysosomal acid α-1,4-glucosidase. We aimed to identify genetic variations and clinical features in Spanish subjects to establish genotype-phenotype correlation. Methods: A total of 2709 subjects who showed symptoms or susceptible signs of PD were enrolled in this observational study. Enzymatic activity was detected by uorometric techniques and the genetic study was carried out using Next-Generation Sequencing. Results: Fourteen different variants from seventeen patients were identied, four of whom had not been described in the literature previously, including a homozygous variant. In all of them α-glucosidase activity was decreased. Muscle weakness, respiratory distress, exercise intolerance, hypotonia, dysphagia and myalgia were commonly observed in patients. Conclusions: This study report four new mutations that contribute to the mutation spectrum of the GAA gene. We conrm that patients in Spain have a characteristic prole of a European population, with c.-32-13T> G being the most prevalent variant. Furthermore, it was conrmed that the c.236_246delCCACACAGTGC mutation in homozygosis is associated with early disease and a worse prognosis.


Background
Pompe disease (PD) is an autosomal recessive metabolic disorder caused by mutations in the acid alpha-glucosidase gene (GAA) that produces biochemical defects in the lysosomal acid alpha 1,4-glucosidase. The de cient activity of the enzyme leads to lysosomal accumulation of glycogen in all tissues, specially skeletal muscle. PD is a disorder that manifests a clinical spectrum that varies regarding the age of onset, the rate of disease progression, and the degree of organ involvement; and in general, there is an inverse correlation between the severity of the disease and the level of residual enzyme activity. Because of the variation in the phenotypes, PD is classi ed into infantile-onset Pompe disease (IOPD) and late-onset Pompe disease (LOPD). IOPD is characterized by onset during the rst year of life with hypertrophic cardiomyopathy, generalized muscle weakness, hepatomegaly and respiratory dysfunction. LOPD is characterized by onset in childhood or adulthood and the presence of slow progressive muscle weakness, cardiomyopathy and respiratory distress. The incidence of the disease varies in different ethnic groups and for different clinical forms. It was reported that the incidence of PD in Caucasian is 1:100000 and 1:60000 in IOPD and LOPD respectively [1] The GAA gene (OMIN 606800) is situated on chromosome 17 and contains 20 exons and 19 introns extended over a distance of 20Kb.
The rst exon is noncoding and the beginning of the start codon is at position 33 of exon 2. To date, over 560 variants have been described in the GAA gene. Most of the variants described are marked as pathological and some of them have uncertain signi cance. Some mutation are widely found in certain populations. For instance, the intronic variant c.-32-13T > G is the most common in Caucasian individuals [2] In Asian population the most common variants are c.1935C > A and c.2238G > C in Taiwanese and Chinese individuals [3], c.1316T > A and c.1857C > G in Korean individuals and c.2560C > T is the most frequent in Afro-American individuals [4] We report here an observational study as a result of a biochemical and genetic analysis of subjects suggestive of PD. We analyzed clinical manifestations, GAA activity and GAA mutations of Spanish patients with Pompe disease to establish genotype-phenotype correlation.

Design of the study and patients
In this observational study, clinical and biochemical aspects and GAA gene sequence in a large cohort of patients from different Spanish hospitals were analyzed.
Patients included either had a family member with PD or presented more than one sign or symptom associated to PD: generalized muscle weakness, CK elevations, exercise intolerance or pain, hipotonia, hypertrophic cardiomyopathy, respiratory distress, dysphagia, dyspnoea. Enzyme activity in DBS [Dried blood spots] was measured for each patient. Patients with decreased enzyme activity in DBS underwent lymphocyte determination to con rm the enzyme diagnosis. The sequencing of the GAA gene was performed in the patients who showed low enzyme activity in DBS and lymphocytes or who had a family member with PD.
Informed consent was signed by all patients and the study was approved by the Ethics and Research Committee of the Virgen Macarena and Virgen del Rocío University Hospital (Code: 0826-N-15).

BioChemical analysis
The determination was carried out according to the technique described by Chamoles et al. [5]. GAA activity was measured in DBS samples or isolated lymphocytes using 4-Methylumbelliferyl-a-D-glucopyranoside as sustrate and acarbose as inhibitor of competing enzymes at pH 4 [6, 7] A standard curve of 4-methylumbelliferon allow to establish a relationship between the intensity of the uorescence and the enzymatic activity as umol/L/h in DBS [cut off < 0,75 umol/L/h] and nmol/min/mg protein in lympohocytes (cut off < 0,15 nmol/min/mg protein).

Molecular and bioinformatics analysis
Genomic DNA was isolated from whole blood by standard procedures using MagNA Pure Compact Nucleic Acid Isolation Kit I. (Roche Diagnostics, Basle, Switzerland). Genetic study was carried out by Next-generation sequencing (NGS). All coding regions and classical splicing sites of GAA gene were ampli ed using a custom design kit for Ion AmpliSeq in a S5 Ion Torrent Platform. The reads were After analyzing the numerous variants obtained, those with a frequency greater than 1% in the general population were discarded according to the polymorphism database [http://www.ncbi.nlm.nih.gov/projects/SNP/]. Variants that were described as benign, probably benign or polymorphisms in databases were not further researched. Novel missense mutation effects were determined using in silico analysis by Mutation Taster [http://www.mutationtaster.org] and Polyphen2 software programs [http://genetics.bwh.harvard.edu/pph2]. Novel nonsense mutation that generates a premature stop codon upstream of another known disease causing nonsense mutation or that affects the active protein center were assessed pathogenic.

Results
From August 2016 to December 2019 were tested 2637 samples of 1343 males and 1294 females [mean age 45.16 years, SD 20.77, range 0-98]. Patients with positive screening in DBS (activity < 0,75 µmol/L/h) are asked for a sample of lymphocytes to measure the enzyme activity and con rm the diagnosis (activity < 0.15 nmol/min/mg protein). The GAA sequence was performed in all patients whose enzyme activity measured in DBS and lymphocytes was decreased (Fig. 1).

Enzymatic GAA activity
Of the 2637 patients studied, 2520 (95,56%) showed normal α-glucosidase activity in DBS. The determination of the enzyme activity in lymphocytes was carried out in the 117 (4,44%) patients who showed low activity in DBS to con rm the enzyme diagnosis. The analysis of the enzyme activity in lymphocytes was normal in 93 (3,53% of total studied and 79,5% of 117 positives in screening) patients and a total of 24 (0,91% of total studied and 20,5 of 117 positives in screening) patients resulted with reduced activity and they were all sequenced.

GAA mutations
In relation to 24 sequenced, 17 subjects (#1 to #17), presented two variants compatible with disease: seven males and nine females with LOPD (mean age 36,07, SD 20,57, range 7-64) and a 2-day-old boy with IOPD. Subjects with two variants found will be called "patients". Subjects #18 and #19, a woman and a man respectively, showed a single variant in heterozygosis (#18 and #19) and in 5 subjects no genetic justi cation was found in this study. All the variants found in the molecular study were consulted in the bibliography and public databases and were subsequently analyzed using in silico tools if it was possible.

Clinical manifestations
Among the 17 patients with PD included in the study, one patient had IOPD phenotype and 16 had LOPD phenotype. The most frequent symptoms and sings in LOPD were muscle weakness (62,5%), followed by high CPK serum values (37,5%) and respiratory distress (25%). Cardiomyopathy, exercise intolerance, hypotonia, dysphagia and myalgia were ascertained in 12.5% of patients. Patient #2, #3 and #6 were asymptomatic at the time of assessment. They were incorporated in the study because they had a member of the family with PD.
The only patient with IOPD, a male of two days of age (patient #1) presented at birth a hypertrophic cardiomyopathy and he died shortly after receiving the sample.
The mean of GAA activity in LOPD patients was 0.30 umol/L/h in DBS and 0.05 nmol/min/mg protein in the isolated lymphocytes.
Enzyme activity in DBS of the only infantile-onset Pompe disease patient was 0.5 umol/L/h and lymphocyte measurement could not be performed due to the death of the patient.

Genotype-Phenotype correlations
Two mutations were the most frequent, contributing to 58% of the total alleles. The most common variant was c.  Table 3].

Discussion
The proportion of patients with PD from the ones on suspection of PD in other similar studies in caucasic population was 0.29% [8] and 2.2% [9] 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 rst 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 rst 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 ndings in Pompe disease studies [14,15]. We con rm 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 [16] 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 con rm 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 signi cance will be explained in the future.
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 [2]. 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][20][21] The next most frequent mutation present in our population was c.236_246delCCACACAGTGC. It was described by Palmer [22] 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 identi ed 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 [23]. 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 identi cation 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 ndings 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 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 [27]. 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 [28] published, in general, mutations in the canonical acceptor and donor sites affect strongly conserved sequences that de ne exon-intron boundaries. Therefore, any variants in these canonical sequences might alter interaction between premRNA and proteins involved in the intron removal.

Conclusions
In this study fourteen genetic variants in GAA gene were identi ed, as cause of Pompe disease, including four new variants. This study con rms that patients in Spain have a characteristic pro le of a European population, with c.-32-13T > G being the most prevalent variant. Furthermore, it was con rmed that the c.236_246delCCACACAGTGC mutation in homozygosity is associated with early disease and a worse prognosis. We propose to extend the genetic study in the 7 individuals without genetic justi cation using techniques that require the study of the intronic zones of the gene or alfa-glucosidase messenger RNA.
Our ndings underscore the importance of early diagnosis and propose to accurate molecular analysis to improve genetic counseling in addition to enabling a better quality of life for patients.

Declarations
Ethics approval and consent to participate    Figure 1 A description of PD screening protocol.

Figure 2
Enzyme activity values in DBS and lymphocytes in patients. Figure 3