Reply to Sabatelli et al.: Detecting Collagen VI in Bethlem Myopathy

Abstract

This is a response to a letter by Sabatelli et al. (1).In their letter, Sabatelli et al. (1) refer to our recent paper on a Bethlem myopathy patient with a homozygous COL6A2 p.D871N mutation. The authors are concerned that our data are inconsistent because we reported that collagen VI staining was absent in muscle when using monoclonal antibody VI-26 but present in very reduced amounts in cultured fibroblast extracellular matrix when detected with monoclonal antibody 3C4 (2). We agree that based on our in vitro fibroblast data it is likely that this patient's muscle contains some collagen VI, and we believe that our staining conditions were not sufficiently sensitive to detect the small amount present even though collagen VI staining was strong in the control muscle. We had this in mind when we said "collagen VI was not detected in UCMD65 muscle" and used the phrase "absent collagen VI staining" under "Results." Unfortunately we used the term "absent collagen VI" in the subheading, and this has resulted in the inadvertent apparent inconsistency. The collagen VI staining was actually done on sections of paraffinembedded muscle, not frozen sections as suggested under "Experimental Procedures," and this is why we used an antigen retrieval protocol, which resulted in strong staining in the control muscle (2). We apologize for omitting this important detail under "Experimental Procedures." Aberrant mitochondria in a Bethlem myopathy patient with a homozygous amino acid substitution that destabilizes the collagen VI ␣2(VI) chain.

Authentication of collagen VI antibodies

“…There has been discussion in the collagen VI literature regarding the use of VI-26/MAB3303 and 3C4 for immunostaining patient muscle tissue and fibroblasts [ 15 , 16 ]. It has been reported that VI-26 is less sensitive at detecting collagen VI in muscle and fibroblast preparations compared to 3C4 leading to an underestimation of the amount of collagen VI degradation [ 23 ].…”

“…Commonly-used antibodies include VI-26 (MAB3303) raised against tissue-purified collagen VI, 3C4/Mab1944 for the α3 chain, and 6A1-H200 for α1. Public discussion within the collagen VI community about the use of antibodies for the molecular and histological analysis of collagen VI mutations [ 15 , 16 ] stimulated an effort to authenticate commonly-used antibodies. In this report we define chain specificity and relative sensitivities of 23 commercially-available collagen VI antibodies by immunohistochemistry and immunoblotting.…”