**4. Discussion**

This study attempted to identify *MT-CYB* mutations in Senegalese ARF patients. Since cardiac tissues were not available for molecular analysis, mutations in this region were assessed in peripheral blood. The present results show a high rate of *MT-CYB* polymorphisms in unoperated subjects (72.73% of mutations). Unoperated ARF patients developed a cardiopathy following ARF; however, since they did not undergo valvular replacement, the disease was seemingly induced in them, and *MT-CYB* mutations were detected in the cells involved in the autoimmune response in blood, similar to T and B lymphocytes, and macrophages, since self-reactive T cells migrate from peripheral blood to the heart and proliferate in the valves in response to stimulation by specific cytokines [6].

Two synonymous substitutions (Y206Y and S193S) associated with ARF (according to the Fisher test) were observed at high frequencies (67.86%) and exclusively in the operated subjects. Despite the absence of amino acid substitutions, the corresponding positions (A15362T and A15323C) may serve as potential molecular markers if consistently present in individuals with ARF. Non-synonymous substitutions T190A, F188I, and I185L, frequent in the unoperated group, are also associated with the pathology. These sites were polymorphic in comparison with the control but monomorphic in comparison with the Caucasian reference sequence. These mutations were detected on MITOMAP but matched with different substitutions. The mutation T15301C (I185L) is associated with idiopathic dilated cardiomyopathy [14] as being a synonymous substitution Leu-Leu. Three missense mutations (S344T, L299F, and I298T), predicted to be pathogenic, have been reported in unoperated patients 2 and 1. The substitution of an aliphatic Leu with an aromatic Phe and that of nonpolar Ile with a polar Thr affected protein structure and function. The substitution of Ser with Thr could have been inconsequential, since they are both thiolated/hydroxylated and polar amino acids and the mutation is homoplasmic (the predictions of this mutation are not formal; thus, mutation may be benign). However, these mutations are in a conserved domain, playing a crucial role in cytochrome b function and thus in the activity of complex III. The C-terminal domain contains the binding sites for ubiquinone/ubiquinol [40,41] and is responsible for proton translocation outside the mitochondrial membrane [42]. Furthermore, the L299F mutation has been reported in breast cancer; however, it was a substitution from cytosine to thymine (according to the reference sequence) at position 15641, whereas the mutation detected herein corresponds to the heteroplasmic substitution of guanine by adenine (according to our control sequence). Both mutations were transitions occurring in the same position. Several mutations were present in both unoperated and operated patients, including G15849A and T15824C synonymous substitutions and the A15784G sense mutation. G15849A and T15824C corresponding to substitutions T368I and T360A have been predicted to be non-pathogenic, although they are polar Thr substitutions by non-polar Ile and Ala. These point mutations occur in a conserved domain in mammals, but are outside the C-terminal domain, thus probably accounting for their benign state. T15824C is present in breast cancer as A15824G, and A15784G has been reported in patients with dilated idiopathic cardiomyopathy [16]. Most (60%) of the mutations were absent in operated patients. Mutations present only in the latter were either synonymous or non-pathogenic, except for those present in one patient (Sg56). More than half of the *MT-CYB* mutations were heteroplasmic (58.23%), probably because polymorphonuclear mitochondrial genomes do not contain *MT-CYB* mutations leading to a mixture of wildtype and mutated genomes or heteroplasmy in immune cells. Cells were not isolated for DNA extraction; either of the hypotheses cannot be considered accurate. However, the current speculation that heteroplasmic mutations are more likely to occur in pathogenic mutations rather than in normal polymorphisms [16] is consistent with the present pathogenic mutations.

Amino acid frequencies did not differ significantly upon pairwise group comparisons. However, Pro and Thr were absent in the controls but present in ARF patients at low frequencies, indeed because of the presence of missense mutations.

Differences in the degree of *MT-CYB* polymorphisms between operated and unoperated patients sugges<sup>t</sup> that valvular replacement suppressed gene mutations. This is corroborated by the results of the genetic diversity of the study population, which facilitated the characterization and analysis of differences between unoperated ARF patients and the other two groups in the study population. Similarly, group comparisons in the study population revealed the genetic proximity of operated subjects and the controls (Pi, k, and the genetic distances were low) and those genetic differences between the operated and unoperated patients. These results may have been obtained because surgery eliminates diseased valvular cells and simultaneously the source of the pathology. The proteins of valvular cells similar to those of *Streptococcus pyogenes* [43] are the targets of immune cells. Eradication of the source of the pathology would lead to a correction between the mechanism underlying autoimmunity, and the cells involved in the defense against *Streptococcus* of Group A and autoimmunity would become functional again in the next generation. Selection analyses have indicated that mutations in the operated subjects follow a neutral evolution, while those present in the unoperated patients are under positive selection. These results are concurrent with previous findings; non-synonymous *MT-CYB* mutations tend to consistently occur in unoperated patients. These variants have a deleterious effect on the protein, and an increase in their frequency would further cause protein damage and simultaneously aggravate the disease. Since these mutations generally do not occur in operated patients and those persisting after valvular replacement are neutral (neither beneficial nor deleterious), the involvement of *MT-CYB* mutations in ARF complications can be considered (RHD).
