Pathophysiology

Pompe disease, or glycogen storage disease type II, is a lysosomal storage disorder and a metabolic myopathy caused by deficiency of lysosomal acid alpha-glucosidase (GAA, also referred to as acid maltase).

GAA hydrolyzes the 1,4 and 1,6 glucosidic bonds of glycogen. This function is required for the breakdown of glycogen into glucose in the lysosomes. Biallelic GAA gene pathogenic variants result into absent or deficient activity of the GAA enzyme, which leads to the accumulation of glycogen in the lysosomes of several cell types and tissues, particularly cardiac, skeletal, and smooth muscle cells. In addition to glycogen storage, a typical secondary feature of Pompe disease pathology is the accumulation of autophagic material in muscle fibers [1, 2]. Normal intracellular metabolism becomes disturbed, including mitochondrial function with oxidative stress activation [3, 4], and/or cytoplasmic glycogen metabolism impairment. Disruption of lysosomes by itself, with release of proteolytic enzymes into the cytoplasm, may also play a role in the disease pathophysiology [5].

Genetics

Pompe disease is inherited in an autosomal recessive manner and is due to biallelic pathogenic variants in the GAA gene. The GAA gene is localized on chromosome 17 at the 17q25.2–q25.3 locus and contains 20 exons including the 19 coding ones [6, 7].

There is a high allelic heterogeneity/diversity: missense, nonsense, splice-site variants, partial deletions, and insertions have been reported to be causative of the disease. As of December 2020, Pompe disease GAA variant database at the www.pompecenter.nl website included 648 disease‐associated variants, 26 variants from newborn screening, and 237 variants with unknown severity [8]. The database is also directly accessible via www.pompevariantdatabase.nl.

The most common pathogenic variant is the intronic mutation c.-32-13T > G (found at the heterozygous state in approximately 80–90% of adult patients and 50% of children) and associated with a slowly progressive course of disease [7]. This splice site mutation results into variable levels of residual activity (up to 20% of normal) and mostly combines in adults with a very severe pathogenic variant on the second allele [8]. Genetic modifiers explaining the broad clinical variability in patients carrying the c.-32-13T > G variant have been identified. For example, the silent, cis-acting c.510C > T variant reduces leaky wild type splicing and thereby residual GAA activity [9]. Patients homozygous for the c.-32-13T > G variant rarely express symptoms [10].

Other relatively prevalent mutations show typical ethnical distribution, such as the p.Glu176Argfs*45 (often referred to as c.525del), p.Gly828_Asn882del, and p.Gly309Arg in the Dutch population, p.Arg854* in Africa, p.Asp645Glu in Taiwan, p.Ser529Val, p.Arg672*, p.Arg600Cys in Japan, p.Trp746Cys in China, p.Gly828_Asn882del in Canada [11, 12].

GAA variants associated pseudo-deficiency of GAA have been described, for example, the variants p.Gly576Ser and p.Glu689Lys, often present in cis [13]. Patients homozygous for these mutations have low levels of GAA activity but do not develop clinical signs of the disease.

Most GAA variants lead to production of some (active or inactive) GAA protein. Patients expressing these variants are called CRIM (Cross Reactive Immune Material) positive. About one third of infantile Pompe disease patients, depending on their genotype, do not express any GAA protein and are defined CRIM negative. For example, the mutation p.Glu176Argfs*45 is a CRIM negative GAA gene variant. CRIM negative patients have a higher risk of producing antibodies against recombinant enzymes when treated with enzyme replacement therapy (ERT) [14, 15].

Frequency

The estimated incidence of Pompe disease has been reported to vary between 1:40,000 and 1:146,000 [7, 16]. Newborn screening programs implemented in some countries have led to reports of figures between 1:8684 and 1:23,596 [17,18,19]. Recent studies have revealed a similar incidence in some European countries [20, 21].

The incidence rate is higher in specific countries and ethnic groups, such as Taiwan (1 in 17,000) [19] and French Guiana (1 in 2000) [22].

Classification

Traditionally, different clinical forms of the disease, outlined in Table 1, have been described in the literature depending on age at onset and severity:

1.

Infantile-onset Pompe disease.

2.

Late-onset Pompe disease or non-classic Pompe disease (childhood, juvenile, adult-onset).

Table 1 Pompe disease spectrum of manifestations

However, the clinical spectrum of Pompe disease is broad and continuous, and symptoms can manifest at any age from infancy to late adulthood.

Skeletal muscle weakness dominates the clinical picture and affects both respiration (including the diaphragm) and mobility. The course of the condition is variable in older children and adults, but it remains relentlessly progressive, resulting in significant morbidity and often in premature mortality. Respiratory failure is the major cause of death [5].

Manifestations and clinical approach
1.

Infantile-onset Pompe disease (IOPD).

The classic infantile form is the best delineated form of Pompe disease and at the most severe end of the clinical spectrum. The disease may be present at birth or within the first few months of life with hypotonia, feeding difficulties or respiratory problems. A hypertrophic cardiomyopathy is characteristically present and may already develop in utero. Without therapy the disease progresses fast, and patients do not achieve major motor milestones like sitting, standing or walking and die within the first year of life of cardiorespiratory failure.

Atypical infantile Pompe disease

Rarely patients with infantile Pompe disease present later (beyond 6 months of age). This atypical form of infantile Pompe disease should be suspected in infants that present within the first two years of life with generalized hypotonia, cardiac hypertrophy, mild liver enlargement, recurrent respiratory infections (due to cardiac disease and hypotonia/weakness of respiratory muscles), macroglossia. Cardiac hypertrophy is mostly less prominent than in the classic form. Development of motor milestones is delayed. Some of these children achieve the ability to sit or stand without therapy.

Together the classic infantile form and the atypical infantile form are frequently named infantile onset Pompe disease. Since patients with the atypical form have a better prognosis, it is important to make the differentiation.

ERT has changed the prospects of patients with infantile Pompe disease dramatically. Overall survival has increased, particularly in children with high-dosage treatment regimens (see also section “Therapy”). Many children learn to walk. However, children are not cured. A new phenotype has emerged in long-term surviving patients.

2.

Late-onset Pompe disease (LOPD)

The phenotype of late-onset Pompe disease is extremely broad and is generally associated with slower disease progression [23, 24]. Patients may present at any age, but mostly after the age of 1 year during childhood or adulthood. They usually present with proximal (limb girdle) myopathy leading to progressive motor disability (more closely related to disease duration than to the age of the patient), with waddling gait, mostly without cardiac involvement. Respiratory muscle involvement may occur early in the course of the disease. Due to the involvement of diaphragm, pulmonary function in supine position may be more affected than in upright position. Respiratory involvement can be accompanied by headache, somnolence, and/or dyspnea. Respiratory and motor involvement do not necessarily have to progress at the same rate. Rarely patients present with respiratory failure.

Smooth muscles may be involved with, as an example, dolichoectasia of cerebral vessels, but only a very few cases have been described in which an aneurysm has led to intracerebral hemorrhage.

Mild myopathic features, creatine kinase (CK) levels < 1000 U/L in adults and up to 2500 IU/l in childhood onset patients and proximal limb girdle weakness and/or axial muscle weakness with or without reduced pulmonary function, in particular when in supine position should be considered as red flags for LOPD patients [25].

DiagnosisNewborn screening

Newborn screening (NBS) for Pompe disease is possible by measuring GAA activity in dried blood spots with different methods (tandem-mass spectrometry, fluorometry, microfluidics) [17,18,19,20,21, 26]. Newborn screening is essential for timely identification and treatment of patients with the infantile-onset forms of the disease.

Targeted next generation sequencing (NGS) could provide additional information and confirmation of the diagnosis for people identified by biochemical screening [27].

However, some limitations of the newborn screening should be considered. First, the assay in dried blood spots is only a screening test and is not sufficient for definitive diagnosis. Second, the NBS screening in its current form cannot discern IOPD from LOPD. LOPD patients are thereby patients in waiting requiring long term follow-up and monitoring which may create uncertainty and a psychological burden for families [28, 29].

NBS programs are already active in several countries (for example, in the US, Taiwan, Japan, some Italian regions) [19, 20, 27]. Pompe disease was added to the US Recommended Universal Screening Panel (RUSP) in 2015 [30].

GAA enzyme assay

A GAA enzyme assay in dried blood spot assay can be used as a first line test. However, this test is not sufficient for a definitive diagnosis. The diagnosis of Pompe disease should be confirmed by GAA enzyme assay in at least one of the following: peripheral leukocytes/lymphocytes, cultured fibroblasts from skin biopsy, muscle biopsy. Common biochemical assays are based on the use of the artificial fluorogenic substrate 4-methylumbelliferyl-α-D glucopyranoside (4MUG) [13]. The discovery that acarbose is a selective inhibitor of maltase glucoamylase allows acid alpha-glucosidase to be selectively assayed in white blood cells and dried blood spots [31].

The possibility of GAA pseudo deficiency should be considered for the interpretation of the GAA biochemical assay (see section “Genetics”) [32]. The use of glycogen as natural substrate enhances the resolution between affected and unaffected; however, the GAA2 pseudo deficiency that occurs in the Caucasian population, can be excluded using 4MUG rather than glycogen [13].

GAA residual enzyme activity in general correlates with phenotype severity, with the lowest activities (< 1%) found in classic infantile patients, and activities from 2 to 40% in late-onset attenuated phenotypes [5].

Molecular analysis of the GAA gene

The molecular analysis of the GAA gene should follow the enzyme assay. This test is useful for further diagnostic confirmation and is necessary for the genetic counseling. Variant classification should follow the American College of Medical Genetics and genomics and Association for Molecular Pathology (ACMG-AMP) system of variant classification which includes 5 classes: benign, likely benign, variant of unknown significance (VUS), likely pathogenic, and pathogenic (class 5 providing ultimate proof of pathogenicity, see for guidance www.pompevariantdatabase.nl). In addition, considering current knowledge about genotype–phenotype correlations, molecular analysis of the GAA gene may provide information about prognosis [7, 33].

The combination of a pathological GAA assay and a genetic confirmation represents the gold standard for Pompe disease diagnosis. This approach is supported by expert consensus statements published in the literature [34, 35], with a moderate-high level of evidence.

Recently, NGS approaches have been exploited in cohorts of patients with skeletal muscle diseases and limb-girdle muscle dystrophies and have allowed for identification of misdiagnosed Pompe disease patients [36].

When clinical suspicion is strong and standard procedures are insufficient, additional molecular methods may be required to validate the diagnosis of Pompe disease, such as a generic splice assay (consisting of exon-flanking RT-PCR and exon-internal RT-qPCR), MLPA, minigene analysis, SNP array analysis, and targeted Sanger sequencing [37].

Complementary laboratory tests

Routine blood chemistry usually shows increased serum levels of AST, ALT, CK, LDH.

A rapid and simple complementary test to identify affected subjects is based on the detection in peripheral blood smears of PAS-positive vacuoles in lymphocytes [38].

In patients with infantile Pompe disease it is important to test for cross-reacting immunologic material (CRIM) status of patients through a Western blot analysis or DNA analysis. Studies in multiple cohorts of patients support the concept that CRIM status may be informative as a prognostic factor and as a predictive element of response to ERT since CRIM negative patients are more likely to develop antibodies against GAA [14, 15].

Analysis of some biomarkers, when available, may be performed, for example the brain natriuretic peptide (BNP) or pro-BNP, reflecting improved cardiac function [39]; the urinary glucose tetrasaccharide (Glc4) [40]; specific skeletal muscle-enriched microRNAs [41, 42]; neurofilament light chain [43, 44].

Multidisciplinary evaluations at diagnosis

Clinical multidisciplinary evaluations at the time of diagnosis or as an initial assessment should include:

For infants with classical IOPD (see also Table 2):

Table 2 Infantile onset Pompe disease—follow-up exams and investigations

General

Physical examination.

Growth parameters.

Neuromuscular evaluation

Neurodevelopmental assessment (specifically in infantile patients)

Cardiology

Pneumology and respiratory function tests

Gastrointestinal and nutritional evaluation

Video fluoroscopic swallowing assessment and evaluation for gastro-esophageal reflux to guide management of feeding (oral/gavage feeding).

Liver ultrasound scan.

Nutritional status and nutrient (protein) intake.

Radiology and imaging

Auditory function

Hearing tests including otoacoustic emissions, tympanometry, and brain auditory evoked potentials (ABR/BAEP).

Ophthalmological evaluation

Language, speech, and oromotor function

For patients with LOPD (see also Table 3)

Table 3 Late onset Pompe disease: follow-up exams and investigations

General

Physical examination.

Growth parameters.

Neuromuscular evaluation

Motor and functional assessments. As LOPD patients may present at any age, depending on their age and level of participation: 6-min walking test (6-MWT) (from the age of 2), Muscular force by Medical Research Council (MMT-MRC) (from the age of 5), timed tests, hand-held dynamometry (from the age of ten), patient-reported outcome measures [39].

Needle electromyography (EMG). EMG and peripheral nerve conduction studies are optional and may be considered at diagnosis as a supportive element.

Muscle biopsy (not needed when other biochemical tests are conclusive for the diagnosis).

Cardiology

Pneumology and respiratory function tests

Pulse oximetry.

Spirometry: forced vital capacity (FVC) sitting; FVC supine; Maximun Inspiratory Pressure and Maximum Expiratory Pressure (MIP/MEP) (from the age of 6).

Polysomnography.

Assessment of need for ventilatory support by home ventilation experts (if applicable).

Gastrointestinal and nutritional evaluation

Video fluoroscopic swallowing assessment and evaluation for gastro-esophageal reflux to guide management of feeding (oral/gavage feeding).

Liver ultrasound scan.

Nutritional status and nutrient (protein) intake.

Auditory function

Hearing tests including otoacoustic emissions, tympanometry, and auditory evoked potentials (ABR/BAEP).

Language, speech, and oromotor function

Ophthalmological evaluation

Visual acuity test.

Orthoptic evaluation.

Others

Radiology and imaging

Dual-energy X-ray absorptiometry (DEXA) scan (to screen for osteopenia/osteoporosis) in adult patients.

Skeletal X-ray in the presence of skeletal dysmorphisms.

Additional evaluations for both IOPD and LOPD

There are several additional imaging techniques that may be available in centers with expertise in the management of Pompe disease and may be advisable to perform both in IOPD and LOPD patients. Even though these tests may be of help in the assessment and evaluation of patient clinical conditions, they require specific experience and skills, and should not be considered as routine or indispensable procedures. These include:

B-mode ultrasound to assess diaphragm thickness and search for diaphragm paralysis and computed tomography (CT) scan for evaluation of lungs and diaphragm thickness.

Magnetic Resonance Imaging (MRI). If compatible with patients’ conditions (the supine position might be associated with aggravated respiratory failure) and with the need for sedation, brain MRI may provide useful information on:

Respiratory muscles, position, and thickness of the diaphragm [45].

Skeletal muscle trophism and fatty degeneration. Whole-body MRI protocols are more inclusive than standard MRI protocols focusing on specific anatomical regions (e.g., paraspinal muscles, tongues, pelvis, thigh), enabling evaluation of relevant muscle groups beyond the pelvis and proximal lower extremities [46].

Brain involvement (in infants compatibly with patient conditions). Recent evidence indicates that classic infantile patients may show white matter abnormalities [47]. So far, they have not been encountered in patients with the atypical infantile form. As these manifestations are not present until later in life, a brain MRI may not be required at the first assessment.

In LOPD patients cerebrovascular manifestations (e.g., aneurysms, vertebrobasilar dolichoectasia, dilatative arteriopathy) have been reported [48].

For most of the basic evaluations there is sufficient support and good quality evidence in the selected literature. The level of agreement on their importance for an accurate assessment of patients’ status is high.

For additional evaluations the indications are somehow less stringent, probably because of a lower number of studies or because some aspects of the disease have been identified only in relatively recent years (for example central nervous system involvement in IOPD patients); thus, the level of evidence in the literature can be assessed as moderate-high.

Differential diagnosis

Depending on the clinical form, differential diagnosis with other disease entities should be considered (Table 4).

Table 4 Differential diagnosisTherapyTherapeutic goals

The therapeutic goals in Pompe disease are:

Infants

1.

Improving survival.

2.

Improving or normalizing cardiorespiratory function.

3.

Improving or preserving normal motor skill acquisitions.

4.

Normalizing growth.

5.

Preventing need for ventilator support.

Late-onset patients

1.

Reducing or stabilizing musculoskeletal damage in symptomatic patients.

2.

Improving or stabilizing respiratory function.

3.

Improving the nutritional state of the patient.

4.

Preventing skeletal dysmorphisms (particularly kyphoscoliosis).

5.

Improving quality of life.

Enzyme replacement therapy (ERT) with recombinant human GAA (rhGAA). (Table 5).

Table 5 Enzyme replacement therapy for Pompe disease

The rhGAA preparation Alglucosidase alfa was approved for the treatment of Pompe disease in 2006 and most of the experience gathered on the efficacy of ERT in Pompe disease has been obtained with this preparation. Alglucosidase alfa has been shown to be effective in improving or stabilizing the disease course both in infantile-onset and in late-onset patients [49,50,51,52,53,54]. The level of evidence on the effects of ERT both in infantile-onset and late-onset Pompe disease patients is based on long term, high-quality clinical studies in large numbers of patients. The level of evidence is high.

Two other rhGAA preparations, both enriched in their mannose-6-phosphate content and with improved muscle-targeting properties, were granted approval in recent years [55,