Introduction

Pompe disease, glycogen storage disorder type II, is a rare multisystem disease occurring in approximately 1 in 40,000 births and with symptoms presenting anywhere from infancy to adulthood. Characteristic disease complications include progressive cardiac and respiratory dysfunction, and skeletal muscle weakness. Alterations of the GAA gene that encodes the information for production of the acid alpha-glucosidase results in a shortage of the enzyme which is needed to convert glycogen to glucose in the lysosome. This leads to build-up of glycogen in muscle tissue and ultimately results in progressive tissue dysfunction over time. In this project we built upon recent work (Baligand, et al.) by characterized 13C MR spectroscopy in both wild type and diseased mice (GAA KO mouse) to evaluate its potential for use in Pompe disease clinical trials, where there is a lack of non-invasive and predictive biomarkers.

Methods

We sought to measure the disease specificity, dynamic range and correlation with standard measures for several MRS biomarkers in diseased vs wildtype mice. This included test/re-test reproducibility measurements as well as correlation of the 13C MRS biomarkers with biochemical tissue glycogen measurements. 13C MRS was performed on Gaa-/- KO mice (N=5) and wild type mice (N=8) at 33-36 weeks of age using a Bruker Biospec 11.7T MRI system. A 20mm 13C surface coil was used for detection of naturally abundant glycogen in combination with a 1H volume coil for anatomical reference and proton decoupling. The hind limb muscles were imaged by placing the mice laterally directly over the surface coil, ensuring coverage of the quadriceps and gastrocnemius. 13C spectra were acquired for 1h using a proton decoupled single pulse acquisition with TR = 500ms, acquisition bandwidth = 25kHz with 1024 acquisition points. Proton decoupling was performed using an Mlev6 CPD sequence with decoupling duration and power of 41ms and 15W, respectively. Both SNR and peak area (calculated using Mnova 11) were calculated following a Lorentzian deconvolution. A 200mM 1-13C-sodium acetate reference phantom was also included for additional normalization of both SNR and peak area. Repeat imaging was performed 1-3 days after the initial acquisitions to assess the reproducibility of the measurements. Following imaging, mice were euthanized, and the quadriceps and gastrocnemius muscles were harvested and flash frozen for glycogen concentration measurements. Tissues were lysed on a benchtop homogenizer with stainless steel beads in distilled water, and boiled and centrifuged to clear debris. Glycogen measurements were performed fluorometrically using a commercial kit according to manufacturer’s instructions (Biovision K646).

Results

13C MRS was successful in detection of glycogen in both knockout and wild type mice with all calculated parameters (including normalization with acetate standard) showing high reproducibility from subject to subject and high test/re-test reproducibility (see Table). Measurement of relatively low glycogen concentrations in wild type mice were challenging due the low SNR. Despite this, a correlation was observed between SNR and biochemical analysis of tissue glycogen concentration (r=0.83).

Discussion and Conclusions

We have shown that 13C MRS can be used to detect elevated naturally abundant muscle glycogen levels in mice with glycogen storage disorder type II disease, and that measurements are repeatable with reasonable accuracy. Variability is thought to be driven mostly by small variations in sensitivity from the exact positioning of the RF surface coil over the muscles, and additional variability in the accuracy of the peak area calculations for the lower glycogen concentrations in WT mice. MR spectroscopy measurements correlated with biochemical analysis of glycogen concentration in skeletal muscle in wild type mice. These results suggest 13C MRS for detection of naturally abundant glycogen without any 13C labeling could present viable biomarker options for use in early phase Pompe disease clinical trials both for stratification and as secondary response assessment.

Acknowledgements

No acknowledgement found.

References

1. Baligand C., Todd A.G., Lee-McMullen B., Vohra R.S., Byrne B.J., Falk D.J., Walter G.A. 13C/31P MRS Metabolic Biomarkers of Disease Progression and Response to AAV Delivery of hGAA in a Mouse Model of Pompe Disease. MOL THER-METH CLIN D, 7(15), 42–49.