Nucleotide
substitution                                          Analysis
------------   ----------------------------------------------------------------------------------------------------
3580 C->A      This sequence change is located at codon 92 in the functional domain of the ND1 gene and changes a
               highly conserved Proline, a hydrophobic AA, to Threonine, a neutral AA, altering the hydropathy
               index from - 0.42 to - 0.52. Protean predicted a change of protein structure, and SIFT predicted
               that it should affect protein function. This mutation was not found in controls, and PolyPhen
               predicted that it should be probably damaging. It was considered potentially pathologic.

4136 A->G      This sequence change is located at codon 277, outside the functional domain of the ND1 gene, and
               changes a highly conserved Tyrosine, a hydrophilic AA, to Cysteine, a neutral AA, altering the
               hydropathy index from -1.3 to 2.5. Protean predicted change of the protein structure, and SIFT
               predicted that it should affect protein function. This mutation was previously reported in Leber
               hereditary optic neuropathy [4]. It was not found in controls, and PolyPhen predicted that it
               should be possibly damaging. It was considered potentially pathologic.

4370 T->C      This sequence change is located in the anticodon loop of tRNA glutamine and was predicted to change
               the shape of this conserved region. It was not found in local controls but was present in a
               heteroplasmic state in one patient with a level of 65%. It was previously reported in association
               with mitochondrial myopathy [5]. It was considered potentially pathologic.

5089 T->G      This sequence change is located at codon 207 in the transmembrane domain of the ND2 gene. It changes
               a highly conserved Isoleucine, a hydrophobic AA, to Serine, a neutral AA, and alters the hydropathy
               index from -2.16 to -1.57. Protean predicted changes to the protein structure, and SIFT predicted
               that it should affect protein function. This sequence change was not found in controls, and PolyPhen
               predicted it to be probably damaging. It was considered potentially pathologic.

5516 A->C      This sequence change is located in a highly conserved region of the acceptor stem of tRNA
               tryptophan. It was not detected in controls and was not previously reported as a polymorphism. It
               was considered potentially pathologic.

6261 G->A      This sequence variant is located at codon 120 in transmembrane domain of the COI, which forms part
               of the catalytic subunit of the enzyme. It changes a moderately conserved Alanine, a hydrophobic AA,
               to Threonine, a neutral AA, altering the hydropathy index from  -0.72 to -0.44. Protean predicted
               the introduction of a flexible region, and SIFT predicted that it should affect protein function.
               This sequence change was not found in controls, and PolyPhen predicted it to be possibly damaging.
               It was reported in lymphocytes of six patients with prostate cancer, where elevated levels of
               reactive oxygen species implied possible pathogenicity [6]. It was considered potentially pathologic.

6880 C->A      This sequence variant is located at codon 326 in COI. It changes a highly conserved Threonine,
               a neutral AA, to Lysine, a hydrophilic AA, and alters the hydropathy index from -0.29 to -0.07.
               Protean predicted a change to the protein structure, and SIFT predicted that it should affect
               protein function. This mutation was not present in controls, but it was heteroplasmic in one patient
               with a heteroplasmy level of 65%. PolyPhen predicted it to be probably damaging. It was considered
               potentially pathologic.

8813 C->A      This sequence variant is located at codon 96, outside the transmembrane domain of ATPase 6 gene. It
               changes a highly conserved Threonine, a neutral AA, to Asparagine, another neutral AA, and alters
               the hydropathy index from -0.06 to 0.26. Protean predicted a change to the structure of the protein,
               and SIFT predicted that it should affect protein function. Its heteroplasmy level was 50%, which is close
               to the accepted threshold of 60% [7]. This sequence change was absent in controls, and PolyPhen
               predicted it to be probably damaging. Given these factors, it was considered potentially pathologic.

9104 T->C      This sequence variant is located at codon 193, outside the transmembrane domain of ATPase 6 gene. It
               changes a highly conserved Phenylalanine, a hydrophobic AA, to Serine, a neutral AA, which alters
               the hydropathy index from -30 to -2.60. Protean predicted no change to the secondary structure of the
               protein, and SIFT predicted that it should not affect protein function. This sequence change was
               absent in controls, but PolyPhen predicted it to be benign. It was considered non-pathologic.

9300 G->A      This sequence variant is located at AA position 32 in the transmembrane domain of COIII
               gene. It changes a moderately conserved Alanine, a hydrophobic AA, to Threonine, a neutral AA, which
               alters the hydropathy index from -0.48 to -0.20. Protean predicted no change in the protein
               structure, and SIFT predicted that it should not affect protein function. This sequence change was
               found in 1.2% of controls, and PolyPhen predicted it to be Benign. It was considered non-pathologic.

9904 T->G      This sequence variant is located at AA position 233 in the outside transmembrane domain of
               COIII gene. It changes a highly conserved Phenylalanine, a hydrophobic AA, to Cysteine, a neutral
               AA, which changes the hydropathy index from 0.84 to 0.88. Protean predicted a change in protein
               structure, and SIFT predicted that it should affect protein function. This sequence alteration was
               absent in controls, and PolyPhen predicted it to be probably damaging. It was considered potentially
               pathologic.

9948 G->A      This sequence variant is located at AA position 248 of the transmembrane domain of the COIII
               gene and changes a highly conserved Valine, a hydrophobic AA, to Isoleucine, a hydrophobic AA.
               Protean predicted a change in the protein structure, and SIFT predicted that it should affect
               protein function. This mutation was previously reported as a somatic mutation in tissues from a
               patient with papillary thyroid carcinoma [8]. It was not found in controls, and PolyPhen predicted
               it to be probably damaging. It was considered potentially pathologic.

10946 A->G      This sequence variant is located at AA position 63 in the transmembrane domain of the ND4
               gene and changes a moderately conserved Threonine, a neutral AA, to Alanine, a hydrophobic AA,
               altering the hydropathy index from -0.58 to -0.86. Protean predicted no change in the protein
               structure, and SIFT predicted that it should not affect protein function. This sequence variant was
               not found in controls, and PolyPhen predicted it to be benign. It was considered non-pathologic.

11865 T->G     This sequence variant is located at AA position 369 in the transmembrane functional domain
               of ND4 gene. It changes a moderately conserved Leucine, a hydrophobic AA, to Tryptophan, another
               hydrophobic AA, altering the hydropathy index from 0.84 to 0.88. Protean predicted a change in
               protein structure, and SIFT predicted that it should affect protein function. This sequence
               alteration was absent in controls, and PolyPhen predicted it to be probably damaging. It was
               considered potentially pathologic.

13253 C->G     This sequence variant is located at AA position 306 in transmembrane functional domain of
               ND5 gene. It changes a highly conserved Threonine, a neutral AA, to Serine, another neutral AA,
               altering the hydropathy index from - 0.7 to - 0.8. Protean predicted a change in protein structure,
               and SIFT predicted that it should affect protein function. This sequence alteration was absent in
               controls, and PolyPhen predicted it to be probably damaging. It was considered potentially
               pathologic.

13936 C->A     This sequence variant is located at AA position 534 outside the transmembrane domain of the
               ND5 gene. It changes a highly conserved Histidine, a hydrophilic AA, to Asparagine, a neutral AA,
               altering the hydropathy index from 0.46 to 0.49. Protean predicted a change of protein structure,
               and SIFT predicted that it should affect protein function. This sequence change was absent in
               controls, and PolyPhen predicted it to be probably damaging. It was considered potentially
               pathologic.

14516 A->G     This sequence variant is located at AA position 53 in the transmembrane domain of the ND6
               gene. It changes a highly conserved Leucine, a hydrophobic AA, to Serine, a neutral AA, altering the
               hydropathy index from -1.81 to -1.36. Protean predicted a change of protein structure, and SIFT
               predicted that it should affect protein function. This sequence change was absent in controls, and
               PolyPhen predicted it to be Probably Damaging. It was considered potentially pathologic.

14525 T->G     This sequence variant is located at AA position 50, outside the transmembrane domain of the
               ND6 gene. It changed a moderately conserved Tyrosine, a hydrophilic AA, with Serine, a neutral AA,
               altering hydropathy index from -0.83 to -0.38. Protean predicted significant changes to the protein
               structure, and SIFT predicted that it should affect protein function. This sequence alteration was
               absent in controls, and PolyPhen predicted it to be probably damaging. It was considered potentially
               pathologic.

14831 G->A     This sequence variant is located at AA position 29, outside the transmembrane domain of the
               CYTB gene. It changed a moderately conserved Alanine, a hydrophobic AA, to Threonine,
               a neutral AA, altering the hydropathy index from 0.14 to 0.42. Protean predicted no change in
               protein structure, and SIFT predicted that it should not affect protein function. This sequence
               change was found in 1.9% of controls, and PolyPhen predicted it to be Benign. This sequence variant
               was reported in the setting of Leber hereditary optic neuropathy [9] but is listed on the Mitomap
               database as a polymorphism. It was considered non-pathologic.

15674 T->C     This sequence variant is located at AA position 310 in the C-terminal domain of CYTB, where
               a pathologic mutation may affect ubiquinol/ubiquinone binding activity. It changes a highly
               conserved Serine, a neutral AA, to Proline, a hydrophobic AA, altering the hydropathy index from
               0.61 to 0.70. Protean predicted no change to the protein structure, and SIFT predicted that it
               should affect protein function. This sequence alteration was absent in controls, and PolyPhen
               predicted it to be possibly damaging. It was considered potentially pathologic.

15924 A->G     This sequence variant is located in the anticodon loop of tRNA threonine, a highly conserved
               nucleotide region. Although, this sequence change was not detected in controls, it is now thought to
               be a haplogroup specific polymorphism [10]. It was considered non-pathologic.

15968 T->C     This sequence variant is located at the D-stem loop of tRNA proline, a highly conserved region. It
               was not detected in controls or reported previously as a polymorphism. It was considered potentially
               pathologic.