Clinical, genetic and functional perspectives on ATP-binding cassette subfamily B member 4 variants in five cholestasis adults

Table 2 Analysis of ATP-binding cassette subfamily B member 4 variants and corresponding pathogenicity in five patients

No.	Zygosity of variant	Nucleotide change	Amino acid change	Reference sequence	Location	Domin	Minor allele frequency	SIFT	PolyPhen-2 (HumDiv)	MutPred-2	NNSplice	Mutation Taster	VASOR	Classification according to ACMG
1	Composite heterozygosity	c.2362C>T, c.2777C>T	p.R788W, p.P926 L	NM_000443.3	Exon20, Exon22	IC4, IC5	2.48e-6, -	0.00, 0.01	1, 0.961	0.904, 0.617	-, -	-, -	0.851, 0.876	Uncertain significance (PS3 + PP3), Uncertain significance (PM2 + PP3)
2	Heterozygosity	c.2362C>T, c.537-32G>T	p.R788W, -	NM_000443.3	Exon20, Intron6	IC4, NA	2.48e-6, -	0.00, -	1, -	0.904, -	-, splicing donor loss	-, Pathogenic	0.851, -	Uncertain significance (PS3 + PP3), Uncertain significance (PM2 + PP3)
3	Heterozygosity	c.1865G>A	p.G622E	NM_000443.3	Exon15	IC3	1.24e-6	0.00	0.921	0.83	-	-	0.915	Uncertain significance (PP3)
4	Heterozygosity	c.1757T>A	p.V586E	NM_000443.3	Exon15	NBD1	-	0.02	0.999	0.873	-	-	0.757	Uncertain significance (PM2 + PP3)
5	Heterozygosity	c.3250C>T, c.C504T	p.R1084W, p.N168N	NM_000443.3	Exon25, Exon6	NBD2, Cytoplasmic	3.10e-6, 0.475	0.00, -	1, -	0.807, -	-, -	-, polymorphism	0.857, -	Uncertain significance (PM2 + PP3), Benign (BA1 + BP4 + BP7)

Minor allele frequency is sourced from gnomAD Database, with samples from at least 25 different countries/regions, mainly from Europe and South Asia. SIFT: A predicted score of less than or equal to 0.05 indicates that the mutation will affect protein function, while a score greater than 0.05 indicates that the effect on the protein is tolerable. PolyPhen-2: The range of predicted values is from 0 to 1. If > 0.5, the mutation is predicted to be harmful, and if < 0.5, it is predicted to be benign. MutPred-2: The general score indicates the pathogenicity of a given variant, ranging from 0 to 1. The higher the score, the greater the pathogenicity tendency. Generally, a score greater than 0.5 is considered a pathogenic mutation. “-”does not affect amino acids or the software is not suitable for this type of mutation. Vasor: It gives out a probability of pathogenicity, which can range from 0 (likely benign) to 1 (likely pathogenic). NBD1: Nucleotide binding domain; TMD: Transmembrane domain; IC: Intracytoplasmic.