Transcript, protein, metabolite and cellular studies in skin fibroblasts demonstrate variable pathogenic impacts of NPC1 mutations

Musalkova, Dita; Majer, Filip; Kuchar, Ladislav; Luksan, Ondrej; Asfaw, Befekadu; Vlaskova, Hana; Storkanova, Gabriela; Reboun, Martin; Poupetova, Helena; Jahnova, Helena; Hulkova, Helena; Ledvinova, Jana; Dvorakova, Lenka; Sikora, Jakub; Jirsa, Milan; Vanier, Marie T.; Hrebicek, Martin

doi:10.1186/s13023-020-01360-5

Orphanet Journal of Rare Diseases

Table 1 Cohort of NP-C1 patients stratified by NP-C clinical phenotypes and corresponding genotypes and results of the cell analyses

From: Transcript, protein, metabolite and cellular studies in skin fibroblasts demonstrate variable pathogenic impacts of NPC1 mutations

Patient No.	NP-C Clinical Phenotype	Genotype Deduced NPC1 protein change (ref. NP_000262.2)	NPC1 Promoter haplotype	Allele expression ratio (%)	NPC1 mRNA level (qPCR)	Relative NPC1 protein amount by Western blot	Colocalization object Pearson’s coefficient	UC/CE ratio	Direct Filipin fluorescence vs. control	LDL-induced Cholesterol esterification rate	Evaluation of vesicular cholesterol by diagnostic filipin test	“NP-C Biochemical Profile”
1	Early infantile	p.[*A605Cfs2];[A1187Rfs55*]	1/2	10/90	0.29 ± 0.15	0.00 ± 0.00	0.07	5.8 ± 0.6	3.0 ± 0.8	< 10	strong	CLA
2	Early infantile	p.[N916del];[*P1245Rfs12**]	1/1	50/50	0.56 ± 0.04	0.05 ± 0.00	0.17	8.1 ± 1.7	2.6 ± 1.0	< 10	massive	CLA
3	Early infantile	p.[I1061T]; [T1176_S1196del;S1197_V1198ins15]^$	1/1	63/37	1.26 ± 0.81	0.09 ± 0.01	0.12	7.0 ± 0.6	2.3 ± 0.9	–	–	–
4	Early infantile	p.[R1186H];[T1205K]	1/4	54/46	0.49 ± 0.21	0.04 ± 0.00	0.07	15.5 ± 1.0	2.7 ± 0.5	< 10	massive	CLA
5 Sb	Late infantile	p.[Y276H];[R1186H]	1/1	48/52	0.87 ± 0.42	0.08 ± 0.01	0.28	10.2 ± 1.5	1.7 ± 0.2	< 10	massive	CLA
6 Sb	Late infantile	p.[Y276H];[R1186H]	1/1	49/51	0.66 ± 0.45	0.14 ± 0.03	0.24	7.7 ± 0.8	1.5 ± 0.5	< 10	massive	CLA
7	Late infantile	p.[R1186H];[R1186H]	1/4	–	0.51 ± 0.31	0.03 ± 0.00	0.15	11.9 ± 1.8	3.0 ± 0.8	–	–	–
8	Late infantile	p.[R1186H];[R1186H]	1/1	–	1.40 ± 0.42	0.05 ± 0.01	0.27	7.1 ± 1.3	2.1 ± 0.8	–	–	–
9	Late infantile	p.[P1007A];[R1186H]	1/1	49/51	0.92 ± 0.30	0.85 ± 0.08	0.72	3.2 ± 0.7	1.3 ± 0.4	300	important	INT
10	Juvenile	p.[S954L];[R1186H]	1/1	51/49	0.88 ± 0.14	0.18 ± 0.01	0.42	3.3 ± 0.6	1.6 ± 0.3	220	strong	CLA
11	Juvenile	p.[S954L];[R1186H]	1/1	55/45	1.60 ± 0.63	0.28 ± 0.01	0.62	3.7 ± 0.9	0.9 ± 0.3	120	massive	CLA
12 Cs	Juvenile	p.[S954L];[R1186H]	1/1	51/49	1.44 ± 0.19	0.29 ± 0.05	0.63	2.1 ± 1.0	1.3 ± 0.2	–	–	–
13 Cs	Juvenile	p.[S954L];[R1186H]	1/1	52/48	0.80 ± 0.27	0.36 ± 0.04	0.62	4.3 ± 0.8	1.2 ± 0.3	185	abnormal	INT
14	Juvenile	p.[P474L];[P691L]	1/2	48/52	0.93 ± 0.57	0.33 ± 0.03	0.77	2.7 ± 0.8	0.9 ± 0.4	25	massive	CLA
15	Juvenile	p.[V950G];[P1007A]	1/4	71/29	0.86 ± 0.49	0.32 ± 0.00	0.77	1.5 ± 0.1	0.8 ± 0.2	510	very significant	INT/VAR
16	Juvenile	p.[R411P];[P1007A]	1/4	51/49	1.29 ± 0.75	0.44 ± 0.01	0.79	3.4 ± 0.2	0.9 ± 0.1	1195	moderate but significant	VAR
17	Juvenile	p.[R411P];[S954L]	1/4	51/49	0.52 ± 0.18	0.52 ± 0.06	0.68	4.6 ± 0.9	1.3 ± 0.2	–	–	–
18	Juvenile	p.[L176R];[S954L]	1/1	48/52	0.60 ± 0.32	0.72 ± 0.08	0.75	3.5 ± 0.6	1.5 ± 0.3	320	strong	INT
19	Juvenile	p.[*Q119Vfs8**];[P1007A]	1/1	25/75	1.60 ± 0.67	0.74 ± 0.03	0.67	3.5 ± 0.6	0.6 ± 0.1	580	moderate but significant	VAR
20	Juvenile	p.[*V270Sfs40**];[S954L]	1/1	36/64	0.66 ± 0.25	0.17 ± 0.03	0.58	4.2 ± 0.9	1.2 ± 0.2	–	–	–
21	Juvenile	p.[*E575Rfs17**];[S954L]	1/5	11/89	0.30 ± 0.13	0.25 ± 0.02	0.67	3.1 ± 0.2	1.1 ± 0.3	220	massive	CLA
22	Juvenile	p.[*A605Cfs2**];[S954L]	1/2	11/89	0.30 ± 0.14	0.29 ± 0.03	0.71	3.9 ± 0.1	1.1 ± 0.2	240	marked	INT
23	Adolescent/Adult	p.[*Q905Rfs31**];[S954L]	1/1	10/90	0.25 ± 0.09	0.30 ± 0.03	0.66	2.7 ± 0.5	0.9 ± 0.1	–	–	–
24 Sb	Adolescent/Adult	p.[A927V];[A927V]	2/4	52/48	0.90 ± 0.37	0.49 ± 0.11	0.69	1.4 ± 0.1	1.1 ± 0.4	834	moderate but significant	VAR
25 Sb	Adolescent/Adult	p.[A927V];[A927V]	2/4	–	0.48 ± 0.30	0.73 ± 0.00	0.69	2.1 ± 0.2	1.2 ± 0.3	337	moderate	VAR
26 Sb	Adolescent/Adult	p.[A927V];[A927V]	2/4	50/50	1.11 ± 0.59	0.74 ± 0.01	0.67	3.3 ± 0.1	1.0 ± 0.3	870	moderate but significant	VAR
Control levels (n = 3)		wt		50/50	0.95 (0.35–1.34)	0.94 (0.56–1.27)	0.77 (0.71–0.80)	3.2 (2.3–3.7)	1.00 (0.80–1.33)	2950 ± 1200	absent/very low	normal

Values in “allele expression ratio” are in the order corresponding to the order of alleles as in the column “deduced protein change”. Bold font indicates frameshift mutations. ^$ effect of the splice mutation on protein assumed from cDNA analysis in Ribeiro et al. [28] LDL-induced cholesterol esterification rates are expressed in pmol cholesteryl-[³H]oleate formed/mg protein/4.5 h. A column linking the cell lines to patients of the cohort of reference [3] is available in supplementary Table S1. Abbreviations: CLA classical, INT intermediate, VAR variant [18], UC unesterified cholesterol, CE cholesteryl esters, Sb sibling, Cs cousin

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