Predicting cross-reactive immunological material (CRIM) status in Pompe disease using GAA mutations: lessons learned from 10 years of clinical laboratory testing experience

Abstract

Enzyme replacement therapy (ERT) for Pompe disease using recombinant acid alpha-glucosidase (rhGAA) has resulted in increased survival although the clinical response is variable. Cross-reactive immunological material (CRIM)-negative status has been recognized as a poor prognostic factor. CRIM-negative patients make no GAA protein and develop sustained high antibody titers to ERT that render the treatment ineffective. Antibody titers are generally low for the majority of CRIM-positive patients and there is typically a better clinical outcome. Because immunomodulation has been found to be most effective in CRIM-negative patients prior to, or shortly after, initiation of ERT, knowledge of CRIM status is important before ERT is begun. We have analyzed 243 patients with infantile Pompe disease using a Western blot method for determining CRIM status and using cultured skin fibroblasts. Sixty-one out of 243 (25.1%) patients tested from various ethnic backgrounds were found to be CRIM-negative. We then correlated the CRIM results with GAA gene mutations where available (52 CRIM-negative and 88 CRIM-positive patients). We found that, in most cases, CRIM status can be predicted from GAA mutations, potentially circumventing the need for invasive skin biopsy and time wasted in culturing cells in the future. Continued studies in this area will help to increase the power of GAA gene mutations in predicting CRIM status as well as possibly identifying CRIM-positive patients who are at risk for developing high antibody titers.

Figure 1

Western blot analysis on protein extracts (20ug loaded) from human skin fibroblast and probed with anti-GAA antibody (Top panel= polyclonal rabbit anti-GAA antibody against human placental GAA; Lower panel= monoclonal anti-GAA antibody, GAA1 provided by Genzyme). β-actin was used as a protein loading control. Top panel: Lane 1-Protein molecular weight marker; Lane 2 – CRIM-positive Pompe disease patient; Lanes 3, 5, 7 and 9 – Empty; Lanes 4, 6, and 8 – CRIM-negative patients; Lane 10 – Normal human fibroblasts. Lower panel: Lane 1 – Protein molecular weight marker; Lanes 2 and 3 – Duplicate lanes for CRIM-negative sample; Lanes 4,7 and 9 – Empty; Lanes 5 and 6 – Duplicate lanes for CRIM-positive sample; Lane 8 – CRIM-negative control (known CRIM-negative patient); Lane 10 – Normal human fibroblasts.

Figure 2

Pie chart showing number of alleles identified with different mutations in CRIM-negative patients (see Table I for details).