When a tissue biopsy is performed, it is important to collect a skin biopsy at the same time, to obtain fibroblasts for possible additional biochemical and genetic studies. In addition to the added value of fibroblasts in the diagnostic evaluation of patients, fibroblasts can be stored and retested when desired, e.g., when new diagnostic possibilities emerge in the future. There are several other benefits of fibroblast testing in the diagnostic evaluation of patients with a suspected mitochondrial disorder (Cameron et al.
2004; van den Heuvel et al.
2004). Fibroblast OXPHOS enzyme activities provide information that is helpful for the selection of candidate genes for molecular genetic analysis, even if enzyme activities are normal. For example, in patients with reduced OXPHOS enzymes in muscle and normal enzyme activities in fibroblasts, a genetic defect might be present in one of the nuclear genes causing mtDNA depletion, for example
POLG (de Vries et al.
2007). It should be noted that normal enzyme activities have also been observed in muscle of several
POLG patients (de Vries et al.
2008; Horvath et al.
2006; Van Goethem et al.
2004). Biochemical observations in fibroblasts of patients with mtDNA mutations are variable. The main reason for this is tissue/cell-type-specific differences in heteroplasmy of mtDNA mutations. Although many nuclear defects will give rise to reduced enzyme activities in fibroblasts, there are several exceptions. In addition to
POLG, other nuclear genetic defects causing mtDNA depletion syndromes, such as
DGUOK and
MPV17, often show normal enzyme activities in fibroblasts (Mandel et al.
2001; Spinazzola et al.
2006). The combination of complex I, III, IV, and/or V deficiency in muscle and normal OXPHOS enzymes in fibroblasts is suggestive for involvement of mtDNA depletion, deletions/rearrangements, or point mutations (van den Heuvel et al.
2004). For mutations in two aminoacyl-tRNA synthetase genes,
DARS2 and
RARS2, normal or mild OXPHOS deficiencies in fibroblasts, respectively, have been reported, while for
RARS2, it has been shown that OXPHOS enzyme activities in muscle vary from normal to severely reduced (Edvardson et al.
2007; Scheper et al.
2007). Fibroblast enzyme deficiencies are common in nuclear defects of genes encoding structural components and assembly factors of the OXPHOS system, and also for defects in genes encoding proteins involved in mitochondrial translation (Coenen et al.
2004; Miller et al.
2004; Saada et al.
2007; Smeitink et al.
2006). It should be noted that a limited number of cases have been reported in which a nuclear genetic defect of a structural respiratory chain building block did not result in an enzyme deficiency of the corresponding enzyme, e.g., complex I (Benit et al.
2001), complex II (Taylor et al.
1996), and complex IV (Vesela et al.
2004), although according to the experiences we have had in Nijmegen, these appear to be quite exceptional cases. Occasionally, patients with normal enzyme activities in muscle show reduced enzyme activities in cultured fibroblasts, in particular for complex I and PDHc (unpublished observations). Confirmation of enzyme deficiencies in fibroblasts of the OXPHOS-deficient index case in families in which the pathogenic genetic defect has not (yet) been uncovered, is required before reliable prenatal testing by measuring OXPHOS enzymes in chorionic villi or amniocytes can be performed (Niers et al.
2003). In Nijmegen, prenatal diagnosis is performed by measuring respiratory chain or PDHc enzyme activities in chorionic villi, provided that the diagnostic examination of the index case has revealed a clearly detectable enzyme deficiency in fibroblasts and muscle (or other tissue), and that mtDNA mutations have been excluded in mtDNA from a sample in which the enzyme deficiency has been detected (Niers et al.
2003; Niers et al.
2001). When these prerequisites are met, reliable prenatal diagnosis can be performed. In addition to the diagnostic applications, fibroblasts provide a model system to study the pathogenesis of novel genetic defects, for example by performing complementation experiments. Several assays have been described to examine MEGS activities in cultured fibroblasts. These are based on the same principles as the MEGS assays for fresh muscle. ATP synthesis, oxygen consumption, and substrate oxidation rates can all be measured in fibroblasts (Cameron et al.
2004; Rustin et al.
1994; Wanders et al.
1993). In many of these assays, cells are permeabilized with digitonin, which allows substrates, ADP, and other assay ingredients to reach the mitochondria and enter the matrix via the mitochondrial transporters. In this way, mitochondrial ATP production, substrate oxidation and respiration rates can be examined in a controlled manner, providing additional information on the biochemical phenotype of patients.