Mitochondrial DNA mutations in renal cell carcinomas revealed no general impact on energy metabolism

Abstract

Previously, renal cell carcinoma tissues were reported to display a marked reduction of components of the respiratory chain. To elucidate a possible relationship between tumourigenesis and alterations of oxidative phosphorylation, we screened for mutations of the mitochondrial DNA (mtDNA) in renal carcinoma tissues and patient-matched normal kidney cortex. Seven of the 15 samples investigated revealed at least one somatic heteroplasmic mutation as determined by denaturating HPLC analysis (DHPLC). No homoplasmic somatic mutations were observed. Actually, half of the mutations presented a level of heteroplasmy below 25%, which could be easily overlooked by automated sequence analysis. The somatic mutations included four known D-loop mutations, four so far unreported mutations in ribosomal genes, one synonymous change in the ND4 gene and four nonsynonymous base changes in the ND2, COI, ND5 and ND4L genes. One renal cell carcinoma tissue showed a somatic A3243G mutation, which is a known frequent cause of MELAS syndrome (mitochondrial encephalomyopathy, lactic acidosis, stroke-like episode) and specific compensatory alterations of enzyme activities of the respiratory chain in the tumour tissue. No difference between histopathology and clinical progression compared to the other tumour tissues was observed. In conclusion, the low abundance as well as the frequently observed low level of heteroplasmy of somatic mtDNA mutations indicates that the decreased aerobic energy capacity in tumour tissue seems to be mediated by a general nuclear regulated mechanism.

Figure 1

Analysis of a shift of heteroplasmy in matched tissue pairs. Denaturating HPLC analysis of a PCR fragment (15587–16185) from case 11 at 56.0°C oven temperature (A, B). Denaturating HPLC analysis revealed a heteroplasmy of the kidney tissue of about 40% (A) and of the corresponding renal carcinoma tissue of over 95% (B). Sequence analysis indicates a 16092C/T heteroplasmy in the kidney tissue (C) and the 16092C mutation in the corresponding renal carcinoma (D). Denaturating HPLC analysis of a PCR fragment (8466–8925) from case 12 at 57.8°C oven temperature (E, F). The kidney tissue shows a heteroplasmy of about 50% (E) and the corresponding carcinoma tissue of about 80% (F). Sequence analysis revealed a 8750C/T heteroplasmy in the kidney tissue (G) and the 8750C variation in the corresponding carcinoma tissue (H). Y=C+T.

Figure 2

Mitochondrial DNA analysis of the renal carcinoma with the somatic A3243G mutation. Denaturating HPLC analysis of a PCR fragment (3079–3505) of case 4 at 58°C oven temperature (A–C). The kidney tissue shows a single homoplasmic peak (A), the corresponding renal carcinoma tissue a heteroplasmy over 90% (B) and the mixture of denaturated and reannealed PCR product of kidney and the corresponding tumour tissue resulted in one hetero- and homoduplex peak of the similar height (C). Sequence analysis of the PCR product indicated the wild-type 3243A variant in the unaffected kidney tissue (D), and the 3243G mutation in the corresponding carcinoma tissue (E), as indicated by arrows. Agarose gel analysis of a restriction digestion of a PCR fragment (3118–3332) with HaeIII, which specifically recognizes the 3243G mutation, and two control sites within the PCR fragment yielding two small fragments (F): undigested full-length PCR fragment of 215 base pairs (lane 1); kidney tissue resulting in a 169-base pair fragment (lane 2); carcinoma tissue resulting in a weak 169-base pair fragment of the residual wild-type 3243A variant as well as 72- and 97-base pair fragments, indicating the 3243G mutation (lane 3); 100 bp molecular weight marker (lane 4). R=G+A.

Figure 3

Western blot analysis of VHL protein content in extracts of three matched tissue pairs. Case 3: kidney control tissue (lane 1) and renal carcinoma tissue (lane 2); case 4: kidney control tissue (lane 3) and renal carcinoma tissue with the somatic A3243G mutation (lane 4); case 5: kidney control tissue (lane 5) and renal carcinoma tissue (lane 6); VHL: 24 kDa.