Table 2. In silico predicted residual activity of CYP21A2 stability mutants lacking functional assays.

Variant	Patient’s second mutation (% REA)	Patient’s phenotype	Expected activity of the variant	∆∆G	In silico activity (%)	Reference
p.H38L	NA	NA	?	−1.64	100	26
p.Y47C	NA	NA	?	−1.64	100	27
p.Y59N	NA	CL	0–5	2.54	4.4	28
p.V69L	p.[Q318;.R356] (0) pH62L in cis*	SV	5–60	1.47	22.5	29
p.S113Y	p.V281L (60)	NC	0–60	0.79	63.3	30
p.S113F	NA	NA	?	1.05	42.6	20
p.Q144P	p.P482fs (ND)	SW	0	0.61	83.2	29
p.F164S	p.I172N (2)	SV	0–5	1.37	26.2	29
p.S165P	p.I172N (2)	NC	10–60	4.94	0.1	31
p.T168N	Chimeric gene (0)	NC	10–60	3.41	1.2	32
p.V211L	NA	N?	?	−0.91	100	33
p.E238K	NA	4 SW, 1 NC	0#	−0.33	100	34
p.V249A	NA	N	100	0.45	100	35
p.L261P	c.290-13A/C>G** (0–2)	SW	0	8.95	0.01	36
p.M283L	p.V281L (60)	NC	0–60	−0.3	100	37
p.S301Y	c.290-13A/C>G** (0–2)	NC	10–60	12.31	0.00	38
p.V304E	c.290-13A/C>G** (0–2)	SW	0	2.44	5.1	29
p.V305D	NA	NA	?	0.99	46.6	20
p.F306V	NA	NA	?	1.87	12.2	20
p.L307V	Deletion (0)	NC	10–60	2.8	2.9	30
p.R316L	p.V281L (60)	NC	0–60	0.03	100	30
p.L317M	c.290-13A/C>G** (0–2)	NC	10–60	−0.8	100	39
p.L317V	N	NC§	?	2.42	5.3	40
p.L321P	NA	NA	?	9.84	0.0	20
p.G381S	NA	NA	?	3.51	1.0	20
p.N387K	p. V281L (60)	NC	0–60	7.55	0.0	41
p.F404S	p.F404S (ND)	SW	0	5.34	0.06	42
p.F404L	p.V281L (60)	NC	0–60	2.51	4.6	30
p.T450P	p.T450P (ND)	SW	0	9.35	0.0	42
p.P459H	p.[ClEx6; Q318X; A391T] (0)	SV	2–5	4.44	0.2	43
p.P459S	Chimeric Gene	SV	2–5	2.68	3.6	32
p.P459L	c.290-13A/C>G** (0–2)	SV	0–5	1.29	29.5	29

In silico predicted activities of mutants lacking functional assays were compared with the expected ones. Expected activity was established considering the residual activity of the mutation on the homologous allele and/or the patient’s phenotype when available. In silico enzymatic activities were calculated from the fitting based on the bovine template using the estimated ∆∆G of each of the variants (Table S1). Activities are expressed relative of the wild type protein (100%). *According to functional assays, p.H62L mutation was classified as a mild mutation. Nevertheless, several alleles were described having another mild mutation in cis with decreased enzymatic activities most likely related to the SV form of the disease⁵⁰,⁶⁰. ^**c.290-13A/C>G mutation creates a new acceptor splice site. Patients bearing this mutation have been described presenting either a SW or a SV phenotype⁸. ^#The classification of the expected activity for this variant was based on the fact that 4/5 patients presented a SW phenotype. ^§The patient described by Byounga et al.⁴⁰, disclosed a 17-OHP post ACTH value of 6, 67 ng/mL. According to the current inclusion criteria, patients would be classified as presenting a NC form of the disease when the post ACTH test is at least 10 ng/mL⁶¹. REA: Residual enzymatic activity. NA: Not available, ND: Not determined; N. Normal; NC. Nonclassical; SV: Simple virilizing; SW: Salt wasting; CL: Classical; N: Normal; ClEx6: Cluster Exon 6 mutations. ?: Insufficient data to estimate the expected enzymatic activity.