Gene testing for osteonecrosis of the femoral head in systemic lupus erythematosus using targeted next-generation sequencing: A pilot study

Table 1 Genotype distribution of single nucleotide variants

Sample	Het_SNV	Hom_SNV	Novel_SNV	Het_InDel	Hom_InDel	Novel_InDel
G1	25	22	2	4	0	7
G2	29	11	0	7	0	7
G3	21	9	1	5	0	7
G4	9	14	1	4	0	7
G5	17	12	2	6	0	5
G6	26	15	1	3	0	7
G7	14	24	1	4	0	7
G8	14	11	2	4	0	7
G9	25	21	0	3	0	5
G10	21	9	1	4	0	6
G11	19	25	0	5	1	6
G12	30	12	0	4	0	4
G13	17	14	1	7	0	7
G14	26	12	0	3	0	5
G15	17	14	0	4	0	6
G16	31	11	4	4	0	8
G17	16	19	1	7	0	6
G18	18	24	1	7	0	7
G19	18	22	2	5	0	4
G20	22	13	1	5	0	6
G21	5	17	4	4	0	8
G22	12	11	2	5	0	6
G23	20	26	1	3	1	5
G24	31	14	2	5	0	6
G25	19	13	2	5	0	7
G26	16	20	1	6	0	4
G27	6	16	2	4	0	6
G28	14	24	1	3	0	4
G29	45	8	0	6	0	6
G30	22	13	1	7	0	6
G31	20	23	3	5	0	6
G32	31	15	0	6	0	5
G33	23	13	0	4	0	4
G34	31	7	2	5	0	6
G35	17	13	2	6	0	6
G36	19	10	0	4	0	5
G37	19	25	1	5	1	7
G38	26	20	1	6	0	6
G39	26	13	2	4	0	8
G40	21	10	1	5	0	6
G41	30	12	1	6	0	7
G42	26	17	1	4	0	7
G43	31	14	1	7	0	7
G44	18	13	0	4	0	6
G45	16	21	0	6	0	7
G46	5	17	4	4	0	6
G47	19	25	0	4	1	6
G48	21	9	2	5	0	4
G49	9	14	0	5	0	4

Het_SNV: Heterozygous genotype SNP (annotation by dbSNP); Hom_SNV: Homozygous genotype SNP (annotation by dbSNP); Novel_SNV: Novel SNV (no annotation by dbSNP); Het_InDel: Heterozygous genotype insertion-deletion (InDel) (annotation by dbSNP); Hom_InDel: Homozygous genotype InDel (annotation by dbSNP); Novel_InDel: Novel InDel (no annotation by dbSNP).

Table 2 Low frequency functional mutations

Gene	First priority	SNV count	Sample count	Mutation (0|1|2)	Mutation 1	GENESKY ControlDB SNV Count	GENESKY ControlDB Mutation (0|1|2)	P1	P2	P3	P4
NOS3	Third	1	1	44|1|0	G19	14	203|15|2	0.399	0.3257	1	0.2235
COL2A1	First1	2	4	45|4|0	G34, G42, G6, G4	10	193|26|1	0.6773	0.6202	1	0.4848
CR2	Second	1	1	48|1|0	G2	5	209|11|0	0.6625	0.7007	1	0.7038

CR2: Complement C3d receptor 2; NOS3: Nitric oxide synthase 3; COL2A1: Collagen type II alpha 1 chain; SNV: Single nucleotide variants. First priority: Take the highest priority of SNVs if the mutation is dominant or homozygous; take the lower one from the top two highest riority SNVs if it is a heterozygous recessive pattern. SNV COUNT: The number of loci contained in the gene. Sample count: The number of samples containing mutaed genes. Mutation (0|1|2): The number of mutations; 0 indicates no mutation, 1 indicates one mutation, and 2 represents at least two mutations; Control: GENESKY database containing 220 samples. P1: The P value for the 2 × 3 χ² test of the sample numbers from the case and control groups with 0, 1, and 2 or more mutations, respectively. P2: The P value for the 2 × 3 χ² test of the sample numbers from the case and control groups with 0, 1, and 1 times or more mutations, respectively. P3: The P value for the 2 × 2 χ² test of the sample numbers from the case and control groups with 0 or 1, and 2 times or more mutations, respectively. P4: The P value for the 2 × 2 χ² test of the sample numbers from the case and control groups with 0 or 1, and 1 times or more mutations, respectively. Mutation 1: Only 1 mutation in the gene.

Table 3 Single nucleotide variant information for low frequency functional mutations

Gene	NOS3	COL2A1	COL2A1	CR2
First priority	Third	Second	First1	Second
SNP ID		rs41263847	rs371445823	rs45573035
Ref allele	G	G	C	C
Alt allele	A	A	T	G
Chrs	7	12	12	1
Position	1.51E+08	48377898	48390360	2.08E+08
Strand orientation	+	−	−	+
Gene region	Exonic	Exonic	Exonic	Exonic
Function	Nonsynonymous SNV	Nonsynonymous SNV	Nonsynonymous SNV	Nonsynonymous SNV
SIFT score	0.128	0.24	0.061	0.958
SIFT Score Pred	T	T	T	T
POLYPhen V2 Score	0.845	0.356	0.938	0
POLYPhen V2 Score pred	P	B	P	B
MutationTaster	1	1	1	1
MutationTaster Pred	D	D	D	N
Cadd	3.677832	2.954573	3.523652	−1.25999
Dann	0.999	0.995	0.996	0.129
Eigen	0.1917	−0.0992	0.1764	−1.5893
Kaviar_20150923		0.002186	1.29E-05	0.00066
1000g_chbs		0.0203		0.0254
esp6500		0.000077	0.000077
ExAC03		0.0026	1.65E-05	0.0007
ExAC03_EAS		0.0307	0	0.0097

CR2: Complement C3d receptor 2; NOS3: Nitric oxide synthase 3; COL2A1: Collagen type II alpha 1 chain.

Table 4 Demographics of patients with systemic lupus erythematosus with osteonecrosis of the femoral head

	Patients (n = 49)	G19	G4	G6	G34	G42	G2
Age (yr)	34.1 ± 11.2	36	34	45	36	33	44
Sex (female/male)	45/4	Female	Female	Female	Female	Female	Female
Disease duration of SLE (mo)	61.8 ± 49.8	48	48	204	168	84	72
Disease duration of ONFH (mo)	14.2 ± 18.8	1	2	2	24	1	48
Fever, n (%)	4 (8.2)	N	N	Y	Y	N	N
Skin rashes, n (%)	28 (57.1)	Y	Y	N	N	N	Y
Photosensitivity, n (%)	8 (16.3)	N	Y	N	N	N	N
Raynaud phenomenon, n (%)	11 (22.4)	N	N	Y	N	Y	N
Oral ulcer, n (%)	5 (10.2)	N	N	Y	N	N	N
Arthritis, n (%)	37 (75.5)	Y	N	Y	Y	Y	Y
Polyserositis, n (%)	12 (24.5)	N	Y	N	N	N	N
Interstitial Pneumonia, n (%)	8 (16.3)	N	N	N	N	N	N
Renal disorder, n (%)	21 (42.9)	N	N	N	N	N	Y
Neurological disorder, n (%)	6 (12.2)	N	N	N	N	N	N
Anemia, n (%)	27 (55.1)	N	Y	Y	N	Y	N
Thrombocytopenia, n (%)	6 (12.2)	N	N	N	N	Y	N
Leukopenia, n (%)	8 (16.3)	N	N	Y	N	N	N
dsDNA, n (%)	28 (57.1)	N	N	Y	N	N	N
AnuA, n (%)	25 (51.0)	N	N	Y	N	N	N
Smith, n (%)	19 (38.8)	N	N	N	N	N	N
AHA, n (%)	16 (32.7)	N	N	Y	N	N	N
rRNP, n (%)	8 (16.3)	N	N	Y	N	N	N
ESR (mm/h), n (%)	46 (83.7)	21	16	34	76	24	59
Low C3, n (%)	11 (22.4)	N	N	N	N	N	N
Low C4, n (%)	10 (20.4)	N	N	N	N	N	N
24-hour urine protein n (%)	32 (65.3)	N	N	N	N	N	N
SLEDAI	1-21 (8.9 ± 4.1)	6	4	8	5	5	10

AHA: Antihistone antibody; AnuA: Antinucleosome antibody; C3: Complement 3; C4: Complement 4; dsDNA: Anti-double-stranded DNA antibody; ESR: Erythrocyte sedimentation rate; rRNP: Antiribosome ribonucleoprotein antibody; SLE: Systemic lupus erythematosus; SLEDAI: Systemic lupus erythematosus disease activity index; Smith: Anti-Smith antibody; Y: Yes or positive; N: No or negative. Except where otherwise indicated, values are expressed as the mean ± standard deviation.