It has previously been demonstrated that mitochondrial DNA (mtDNA) mutations within the ND2 gene of histologically normal parotid salivary gland tissue of smokers may be molecular biomarkers for smoking-induced mtDNA damage. Oral squamous cell carcinoma (SCC) is strongly related to cigarette smoking; therefore, we used PCR and direct sequencing to establish whether mtDNA mutations were also present in oral SCC which could be used as additional biomarkers for smoking-associated DNA damage. In addition to searching for mutations in the ND2 gene, the mitochondrial D-Loop was also analysed. Three mutation hotspots were observed in the D-Loop at nt 146, 152 and 186, two of which (nt 146 and 152) have also been implicated in oesophageal SCC, another smoking-related cancer. The mutation hotspot observed at nt 186 has not previously been reported in other tumours. Furthermore, we show that the mutations previously reported within the ND2 gene in normal parotid tissue of smokers were not evident in these samples, but that a mutation hotspot occurs at nucleotide 4917 in oral SCC. We also show that D-Loop mutations occur predominantly in male smokers and female non-smokers and that this association with gender is statistically significant (P = 0.003). We conclude that the mtDNA mutation hotspots found in this study, in particular nt 186, are potential biomarkers for oral SCC. However, owing to gender-specific differences in occurrence in smokers and non-smokers, and a lack of environmental smoking history, in general, it is difficult to associate these mutations with mtDNA damage induced by smoking. If the mutations observed in the subset of male patients are smoking induced, given our previous findings, mutation hotspots in the ND2 gene may be tissue specific suggesting the causative mutagens for mtDNA damage within these tissues are likely to be different.

Mutations in fukutin related protein (FKRP) are responsible for a common group of muscular dystrophies ranging from adult onset limb girdle muscular dystrophies to severe congenital forms with associated structural brain involvement, including Muscle Eye Brain disease. A common feature of these disorders is the variable reduction in the glycosylation of skeletal muscle alpha-dystroglycan. In order to gain insight into the pathogenesis and clinical variability, we have generated two lines of mice, the first containing a missense mutation and a neomycin cassette, FKRP-Neo(Tyr307Asn) and the second containing the FKRP(Tyr307Asn) mutation alone. We have previously associated this missense mutation with a severe muscle-eye-brain phenotype in several families. Homozygote Fkrp-Neo(Tyr307Asn) mice die soon after birth and show a reduction in the laminin-binding epitope of alpha-dystroglycan in muscle, eye and brain, and have reduced levels of FKRP transcript. Homozygous Fkrp(Tyr307Asn) mice showed no discernible phenotype up to 6 months of age, contrary to the severe clinical course observed in patients with the same mutation. These results suggest the generation of a mouse model for FKRP related muscular dystrophy requires a knock-down rather than a knock-in strategy in order to give rise to a disease phenotype.