Mitochondrial Variants in Pompe Disease: A Comparison between
Classic and Non-Classic Forms
Pompe disease (PD) is a progressive neuromuscular disorder that is caused by glucosidase acid alpha (GAA)
deleterious mutations. Mitochondrial involvement is an important contributor to neuromuscular diseases. In this study the
Materials and Methods
In this case-control study, the sequence of
The extent of mitochondrial involvement in the classic group was more significant than in the non-classic
Pompe disease (PD, OMIM #232300) is a progressive myopathy with an autosomal recessive mode of inheritance (1). The combined incidence of PD is generally 1 in 40,000 (2). It has two common forms (early-onset/classic and late-onset/non-classic) with differences in degree of disease severity, age of onset and organ involvement (3, 4). The patients present a broad spectrum of clinical variability such as cardiomyopathy, hypotonia and respiratory insufficiency (5, 6).
They suffer from deficiency or lack of acid alphaglucosidase
According to previous reports, mitochondrial dysfunction
can affect the neuromuscular system (7). Mitochondria (mt)
are essential to aerobic respiration by producing adenosine
triphosphate (ATP). The function of mt is controlled by both
the mtDNA and nuclear genomes, and mtDNA variants may
be affected by nuclear genome variants or vice versa (8). It is
therefore possible that mtDNA genes interact with GAA. To
test this hypothesis,
Materials and Methods
In this case-control study, we recruited 28 PD patients (17
infants and 11 adults) from the Department of Neurology of
both Shariati and Mofid hospitals from December 2013 to
February 2015. In this study, 100 healthy controls were also
recruited comprising 17 infants and 83 adults. An informed
consent was obtained from each participant or a parent in
the case of infants. PD was diagnosed based on clinical
findings by two expert neurologists, measurement of
DNA was extracted from whole blood by using QIAamp DNA Blood Mini Kit (QIAGEN, Germany) according to the manufacturer’s instructions. Quantity and quality of DNA were checked by NanoDrop ND-1000 (NanoDrop Technologies, USA) at 260/280 nm wavelengths and running on an agarose gel (1%) respectively. Total RNAwas extracted from fresh whole blood samples by using the Hybrid-RTM Blood RNA kit and following its protocol (see http://www. tribioscience.com/files/315-150.pdf). RNA concentration and integrity were measured by NanoDrop and agarose gel respectively prior to cDNA synthesis. Presence of sharp bands for both 18S and 28S rRNA was checked. Purified RNA was then stored at -80°C.
Total RNA was used to synthesize cDNA by using the cDNA synthesis kit (Fermentas, Germany) according to manufacturer’s instructions. Briefly, 2 µg of total RNA, 1 µL of oligo dT and random hexamer primers and 8 µL nuclease-free water were mixed in a sterile, nuclease- free tube and placed on ice. After incubation at 65°C for 5 minutes, it was chilled on ice and 4 µL of 5X reaction buffer, 1 µL of RiboLock RNase Inhibitor (20 U/µL), 2 µL of 10 mM dNTP mix, 1 µL of RevertAid M-MuLV RT (200 U/µL) were added.
The mixture was centrifuged briefly and incubated for 5 minutes at 25°C followed by 60 minutes at 42°C. The reaction terminated by heating at 70°C for 5 minutes and stored at -80°C until further use.
The PCR reaction included 50 ng of genomic DNA, 1 µL
of each primer (10 pmol), 0.2 mM of each deoxynucleoside
triphosphate (dNTP), 1.5 mM MgCl2 and 1 U of Taq
polymerase (CinnaGen, Inc, Iran). Cycling conditions for
all PCR reactions were an initial denaturation at 94°C for
4 minutes, followed by 35 cycles of denaturation at 94°C
for 35 seconds, annealing at 56° for 35 seconds, extension
at 72°C for 35 seconds, and a final extension at 72°C for 5
minutes. PCR-amplified fragments were sequenced by
Macrogen (South Korea) using the same PCR primers in
both directions along with a series of overlapping primers to
cover all regions of interest for more accurate results. Finch
TV version 1.4 (Geospiza, Inc., USA) was used to analyze
the chromatograms and were then checked using BLAST
MT-ATP 6/8 expression analysis
Expression levels of
qPCR reactions were performed on a Corbett 6000 PCR-Real-time Detection System with a total volume of 20 µl reaction mixture, containing 1 µl DNA template (50 ng), 10 µl SYBR Green PCR Master Mix (Takara, Japan), 8 µl nuclease- free water and 0.5 µl of each primer (10 pmol). The cycling conditions were an initial denaturation step at 95ºC for 30 seconds followed by 40 cycles of a denaturation step at 95ºC for 12 seconds and an annealing step at 58ºC for 35 seconds. Melting curve analysis was used to validate the specificity and identity of the PCR product for each primer pair. Each sample was run in duplicate to ensure the reliability of the results. Also, a non-template control was included in each qPCR run.
Quantitative variables, in the form of frequency, such as participant characteristics and mitochondrial involvement were described as mean ± SD. Fisher’s exact test was used to compare frequency of mitochondrial involvement in PD and control groups. P<0.05 was considered statistically significant. All analyses were implemented in SPSS version 16 (IBM, USA). Bonferroni’s multiple-testing correction was used to adjust the significance level (a). For frequency comparison of the identified 14 variants, a was set to 0.0036. For differential expression, given that two genes were compared, a was set to 0.025.
Screening of MT-ATP6/8 in patients resulted in the
identification of 14 variants, of which three were novel
variants (Fig .1,). Of the total, 7 variants were in the
classic group, 4 in the non-classic group and 3 were
shared between the two groups (Table 1,). There were
four synonymous and ten non synonymous variants in
|Infant/Adult||Nucleotide||Locus||Amino acid change||R/N.R||Hm/Ht||Pompe||Control||P value|
R and N.R; Denote reported and not-reported respectively, Hm and Ht; Denote homoplasmy and heteroplasmy respectively, and PD; Pompe disease.
|Gene||Nucleotide position||Polyphen-2 score||CADD score||Prediction effect|
MT-ATP6/8 genes expression
A significant decrease was observed for
MT-Cytochrome C oxidase
Screening of MT-Cytochrome
Moreover, based on the analysis of the sequence of lysine tRNA and aspartate tRNA genes, the A8302G variant was found in the former gene in two brothers in a pedigree (Fig .3A,) and the T7572C variant was found in the latter gene in two brothers in another pedigree (Fig .3B,). Variants C15904T and G15928A were also found in two infants in the threonine and tyrosine tRNA genes, respectively.
|- Pedigree diagram. A. Pedigree of a family with a A8302G variant in siblings and B. Pedigree of a family with a T7572C variant in siblings.|
|Infant/Adult||Nucleotide||Locus||Amino acid change||R/N.R||Hm/Ht||Pompe||Control||P value|
R and N.R; Denote reported and not reported, respectively and Hm and Ht; Denotes homoplasmy and heteroplasmy, respectively.
PD is a heterogeneous neuromuscular disorder. Patients
suffer from myopathy, hypotonia and other neuromuscular
manifestations (5). Some tissues such as the nerve and
muscle are more susceptible to mitochondrial dysfunction
since these tissues are highly dependent on oxidative
phosphorylation (10). According to previous studies,
mitochondrial abnormality has been observed in PD
patients (11-21). It is therefore possible that mitochondrial
variants have a secondary role in PD. In this study,
The role of complex V variants has already been seen
in the increase of free radicals. They can affect gene
expression as a secondary factor. In these genes, some
amino acids are conserved and any change could be
potentially pathogenic (23, 24). Fourteen variants were
With respect to G8697A, methionine is a conserved
amino acid. It has a sulfur in its structure that tends
to form beta-sheets and despite owning hydrophobic
properties, it can interact with some electrophilic regions.
In contrast, isoleucine participates in alpha-helix structure
and plays a role in ligand binding to protein. Such a
replacement may change the structure and function of
The C8684T variant in MT-ATP6 changes threonine to isoleucine. The former is a polar amino acid while the latter is non-polar. This change may affect the tertiary structure of the protein and its interaction with the ATP molecule. This variant was also observed in ataxia and autism (29). The C8684T was observed in MS and Huntington’s (23), and G8697A in MS (23) and ataxia telangiectasia (27). The C8562T is a synonymous variant which has been reported in patients with ataxia (29). A8456C and C8406T have benign effects. A8860G was found in 85.71% of patients. In addition, the variants G8697A and A8701G were previously reported in cardiomyopathy. This variant has also been reported in neurodegenerative diseases (30).
It is possible that the cardiomyopathy observed in PD
patients is the result of the presence of these variants, by
Mitochondrial DNA encodes 3 subunits for cytochrome C oxidase. It is the last enzyme in the mitochondrial respiratory chain and is responsible for electron transfer from the cytochrome to oxygen (31). Impairment of cytochrome C oxidase is clinically highly heterogeneous. It starts at any age and includes a diverse range of myopathy to severe multi-organ involvement (32).Genetic defects that affect the structure and function of this gene are usually severe and often lead to fatal metabolic disorders. Such disorders usually occur before the age of 2 and involve tissues such as the heart, muscle and liver, however, its manifestation in adulthood is with less severity. Severity of this disorder can vary even in family members.
In the case of early onset cardiac muscle involvement
is usually associated with hypertrophic cardiomyopathy,
however, in late onset cases, myopathy and hypotonia are
observed (33). It seems that the pattern of manifestations
The observed A8302G variant in lysine tRNA has been previously reported in encephalopathy (35). Also, Govindaraj et al. (36) reported this mutation in three patients with Madras motor neuron disease (MMND).
The variant T7572C located on the T arm of the gene encoding aspartate tRNA was previously reported by Reddy et al. (37) in myelodysplastic syndrome, however, Li et al. (38) found it as a neutral polymorphism, showing that this variant did not affect the secondary structure of the corresponding tRNA by using RNA fold.
G15928A, which is located at the anticodon stem of the tRNAThr, is shown to be a neutral polymorphism (38). The C15904T variant is also shown to be a polymorphism which occurs in the general population with low frequency (39). The C15904T and G15928A variants were observed in patients with encephalopathy by Houshmand et al. (40). Overall, validation of the function of each variant is recommended.
The extent of mitochondrial involvement in the classic group was more severe than the non-classic group. According to these findings, it seems that mtDNA variants have a secondary role in PD. Understandingthe role of mitochondria in the pathogenesis of PD may potentially lead to the development of new therapeutic strategies.
We thank all the participants in this study. This study was funded by Tehran University of Medical Sciences, Tehran, Iran (Grant Number: 23162). The authors declare no conflict of interest.
F.B., S.M.A., M.H., M.H.M.; Conceptualization, investigation, methodology, supervision, validation, visualization, writing-review and editing. F.B.; Data curation. F.B., M.H.; Formal analysis. F.B., S.M.A., M.H.; Project administration. F.B., S.M.A.; Software and writing-original draft. All authors read and approved the final manuscript.