Table 2.

(A) tRNA scoring system implemented in HmtVar according to Yarham and Diroma criteria. The conservation criteria were modified thanks to the availability of Phastcons (31) and Phylop (32). (B) tRNA variant attributes to be considered for future improved disease score estimation

A	tRNA Scoring Criteria	Yes	No	Yes Normalized
	tRNA structure parameters and reports
	Variant described as pathogenic by more than 1 report	2	0	0.1
	Frequency and population data
	PhastCons conservation	1	0	0.05
	PhyloP conservation	1	0	0.05
	Variant Heteroplasmy
	Heteroplasmy evidences	2	0	0.1
	Evidences from functional studies
	Segregation of mutation with disease	2	0	0.1
	Histochemical evidence of mitochondrial disease	2	0	0.1
	Biochemical defect in OXPHOS complexes I, III or IV	2	0	0.1
	Pathogenicity evidence in trans mitochondrial cybrids or mutant mt-tRNA steady state level studies	5	0	0.25
	Evidence of mutation segregation with biochemical defect from single-fiber studies	3	0	0.15
B	Additional tRNA variants Scoring Criteria
	tRNA structure parameters and reports
	Cloverleaf-shaped secondary structure variation
	Post-transcriptional modification
	3D interaction involved in folding
	Frequency and population data
	Allele variant frequency > 2%
	Allele variant frequency in patients > allele variant frequency in healthy individuals
	Macrohaplogroup-defining variant