**3. Results**

#### *3.1. Evaluation of MT-CYB Polymorphisms*

#### 3.1.1. Analysis of MT-CYB Mutations

BLASTn analysis revealed 97% identity with *MT-CYB* and 5 gaps. BLASTx analysis revealed 98% identity (without gaps) with the cytochrome b (mitochondrion) [*Homo sapiens*] [35] and [*Homo sapiens subsp. 'Denisova'*] [36] and 97% identity (without gaps) with cytochrome b (mitochondrion) [*Homo heidelbergensis*] [37] and [*Homo sapiens neanderthalensis*] [38].

Alignment of the chromatograms of ARF patients (operated and unoperated) with those of the controls (Sg97) revealed 165-point mutations with a score of ≥20 or 60 di fferent variants, in the overall study population. Among them, 41.67% corresponded with homoplasmic mutations and 58.23% corresponded with heteroplasmic mutations. Transitions largely surpassed the transversions (respectively 61.67% and 38.33%). Most mutations were present in unoperated ARF patients (72.73%) wherein 7.14 ± 14.70 mutations on average were harbored per individual. Indeed, seven mutations were present at high frequencies in ARF patients (G15849A, T15824C, A15784G, A15362T, A15323C, G15314C, and A15308T). Synonymous (A15362T and A15323C) and missense (G15314C and A15308T) mutations were exclusively present in unoperated ARF patients at frequencies of 67.86%, 67.86%, 42.86%, and 57.14%, respectively. The Fisher test revealed significant results for these mutations, thus indicating their association with ARF. Overall, 36 mutations were absent in the operated ARF patients. Thus, operated ARF patients harbored 1.39 ± 2.97 mutations on average. Di fferences in the average mutation frequencies were significant (p = 0.031). The frequency for synonymous mutations was 38.33% among the ARF patients. Among these amino acid substitutions, nine were pathogenic according to the predictions of Polyphen-2, SIFT, and PROVEAN. All the pathogenic mutations were absent in the operated patients, except for the missense substitutions K287P and N286P, present in all positions of their corresponding codons and present in only one subject (Sg56). Most mutated sites were detected through MITOMAP (https://www.mitomap.org/MITOMAP); however, the corresponding bases di ffered from the those in the present sequences (Table 1).

#### 3.1.2. Variability of Amino Acids

Transcription was carried out based on the second reading frame. Gly and Leu were the most frequent amino acids in the protein. Asn and His were absent in the ARF patients and the controls. Pro and Thr were absent in the controls but present at low frequencies in the ARF patients. However, none of the P-values is significant (Table 2).

#### 3.1.3. Determination of Genetic Diversity of MT-CYB

The analysis of the genetic diversity of the study population is summarized in Table 3. In the study population, 12, 28, and 54 individuals belonged to the control, unoperated, and operated groups, respectively. The sizes of the sequences were the same for each individual (492 bp). The unoperated group di ffered from the other two groups and displayed the highest *MT-CYB* polymorphism frequency. The nucleotide frequencies were similar in all groups. Cytosine was the least represented base with a minimal frequency at the third codon position in contrast with thymine and guanine, which were more frequent at this position. Adenine occurred mostly at the first codon position. In position 1, no transversions occurred, and all mutations were transitions (conservation of the mutated base); hence, the mutation rate (R) tended towards infinity.


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**Table 1.** Characteristics of *MT-CYB* mutations.

#### *J. Cardiovasc. Dev. Dis.* **2019**, *6*, 36


**Table 1.** *Cont.*

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#### *J. Cardiovasc. Dev. Dis.* **2019**, *6*, 36


position according to the protein reference sequence (UniProt accession number: P00156); **CD**: Conserved domain [MT-CYB (52-570) preserved in mammals; Cytochrom\_B\_C (97-399) preserved in cellular organisms; QcrB (97-564) preserved in cellular organisms; cytochrome\_b\_C (115-555) containing the redox sites of quinol and the polypeptide binding site, preserved in cellular organisms; MT-CYB6/f-IV (301-435) preserved in cellular organisms]; **Homo**: Homoplasmic mutation; **Hetero**: Heteroplasmic mutation; **T**: Transition; **t**: Transversion; **(1)**: [14] (A > G); **(2)**: [39] (T > C); **(3)**: [16] (A > G); **NP**: Not pathogenic; **P**: Pathogenic; **P.D**: Probably damaging; **p.D**: Potentially damaging; **TOL**: Tolerable; **Del**: Deleterious; **\***: Significant Fisher; +: Listed in MITOMAP, the letters in parentheses represent the referenced substitutions.

#### *J. Cardiovasc. Dev. Dis.* **2019**, *6*, 36

**Table 1.** *Cont.*

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**Table 2.** Frequencies of amino acids according to populations.

C: Controls, O: Operated, NOP: Unoperated.



3.1.4. Evaluation of the Di fferentiation and Genetic Structuring of MT-CYB in Accordance with the Study Population

The genetic distance was the largest among unoperated ARF patients (0.011 ± 0.002) and the lowest in the controls (0.003 ± 0.001). Genetic distances were greater between unoperated and operated ARF patients (0.009 ± 0.001) than between the controls and operated ARF patients (0.005 ± 0.001) (Table 4).

Analysis of the genetic differentiation factor (Fst) revealed no genetic differentiation of *MT-CYB* between the controls and patients (p = 0.58559). However, a genetic differentiation of *MT-CYB* between operated and unoperated subjects was observed (p = 0.01466) (Table 5). Nevertheless, AMOVA (Table 6) revealed that more than 98% of genetic variability between unoperated and operated ARF patients is of an intra-population origin.





In italics, the p-values and in bold the significant p-value.


#### 3.1.5. Evolution of MT-CYB Mutations

As shown in Table 7, the results of the Z selection test among groups were recorded. The non-significant p-values (0.965, 0.483, and 1.000) among operated ARF patients indicate that their mutations follow a neutral evolution. However, among unoperated ARF patients, the p-value, highly significant under the neutrality hypothesis (0.015), highly significant under the hypothesis of positive selection, and not significant under the hypothesis of negative selection, shows that *MT-CYB* mutations in the unoperated patients are under positive selection.

**Table 7.** Results of the Z selection test (p-values).


In boldface: Significant p-values.
