Immunogenetic Relationship of HLA-G 14 bp Insertion/Deletion Polymorphism and Toll-Like Receptor 9 with Systemic Lupus Erythematosus in Egyptian Patients: A Case-Control Study

Figures & data

Table 1 Demographic Characteristics, Clinical Manifestations, Lab Findings and Disease Activity of SLE Patients

Variable		(n=102)
Age (Years)	Mean ± SD	31.61±11.17
	Median (Range)	29 (18–55)
Disease duration (Years)	Mean ± SD	5.94±2.57
	Median (Range)	5.5 (2–13)
Variable		No	(%)
Onset	Early (≤20 years)	39	38.2
	Late (>20 years)	63	61.8
Manifestations	Malar rash	51	50
	Hair falling	62	60.8
	Photosensitivity	55	53.9
	Oral ulcers	24	23.5
	Discoid lesion	17	16.7
	Arthritis	50	49
	Neurological manifestations (headache)	12	11.8
	Lupus nephritis	54	52.9
	Secondary APLS	11	10.8
Laboratory findings	CBC profile abnormalities	70	68.7
	Leukopenia	12	11.8
	Anemia	53	52
	Thrombocytopenia	5	4.9
	Low C3 level	54	52.9
	Low C4 level	54	52.9
	Positive ANA	102	100
	ANA pattern:
	Homogeneous	52	51
	Speckled	36	35.2
	Nuclear	7	6.9
	Peri-nuclear	7	6.9
	Positive anti-dsDNA	83	81.4
	Positive ACLA	31	30.4
SLEDAI	Mild activity 1–5	29	28.4
	Moderate activity 6–10	31	30.4
	High activity 11–19	30	29.4
	Very high activity ≥ 20	12	11.8
	Mean ± SD	10.31±6.08
	Range	10 (2–22)

Notes: Normal WBCs count =4–11 x 10 ³ /mm³ and leukopenia <4000/ mm³, Normal hemoglobin level=12–16 g/dl and anemia<12g/dl, Normal platelet count=150–400 x 10 ³ /mm³ and thrombocytopenia <100,000/ mm³, Normal C3 level =90– 210 mg/dl, Normal C4 level =13– 75 mg/dl.

Abbreviations: SD, standard deviation; APLS, antiphospholipid syndrome; CBC, complete blood count; C3, complement 3 level; C4, complement 4 level; ANA, antinuclear antibodies; Anti-ds DNA, Anti-double stranded DNA antibodies; ACLA, anticardiolipin antibodies; SLEDAI, systemic lupus erythematosus disease activity index.

Figure 1 Gel electrophoresis of PCR products demonstrating different HLA-G genotypes of the studied participants. Lane L: 100-bp DNA Ladder; lanes 2, 3, 4, 6, 11, 12, 14, 15, 16: HLA-G homozygotes ins/ins (+14bp/+14bp); lanes 9,10: HLA-G heterozygotes ins/del (+14bp/-14bp); lanes 1, 5, 7, 8,13: HLA-G homozygotes del/del (−14bp/-14bp). Depending upon the insertion or deletion of 14 bp sequence in HLA‐G exon 8, PCR products of length 224 or 210 bp were generated, respectively. Three different genotypes homozygous for 14 bp insertion [ins/ins] (+/+14 bp) 224 bp, homozygous for 14 bp deletion [del/del] (−/−14 bp) 210 bp and heterozygous for 14 bp insertion/deletion [ins/del] (+/−14 bp) 224 bp and 210 bp were generated.

Table 2 Genotypes and Allele Frequency of HLA-G Among the Studied Groups

Group Genotypes	SLE Patients (n=102) No (%)	Controls (n=102) No (%)	χ^2	P value	OR
Del/del	28 (27.5%)	55 (53.9%)	Ref	–	1
Ins/del	14 (13.7%)	37 (36.3%)	0.58	0.45	0.74 (0.4–1.6)
Ins/ins	60 (58.8%)	10 (9.8%)	41.99	<0.001**	11.79 (5.25–26.48)
Allele
Del	70 (34.3%)	147 (72.1)	58.4	<0.001**	4.94 (3.24–7.52)
Ins	134 (65.7%)	57 (27.9%)

Note: **Highly significant.

Abbreviations: χ², Chi square test; OR, odds ratio; Del, deletion; Ins, insertion.

Table 3 Relation Between Genotype Frequency of HLA-G and Clinical Manifestations, Laboratory Findings and Disease Activity Among SLE Patients

Variable		Del/Del (n=28)		Del/Ins (n=14)		Ins/Ins (n=60)		Test	P	Post Hoc
Age (Years)	Mean ± SD	32.5±8.9		34.96±10.5		32.5±11.9		2.4^	0.2	–
	Median (Range)	31.5 (20–47)		34 (24–55)		31 (18–53)
Duration (Years)	Mean ± SD	5.8±2.4		5±1.04		6.2±2.9		2.8^#	0.3	–
	Median (Range)	5 (3–10)		5 (4–6)		6 (2–13)
Variable		No	(%)	No	(%)	No	(%)	χ^2	P	Within Groups
Onset	Early (≤20 years)	22	34.9	7	11.1	34	54	4.8	0.09	–
	Late (>20 years)	6	15.4	7	17.9	26	66.7
Manifestations	Malar rash	15	29.4	7	13.7	29	56.9	0.21	0.9	–
	Hair falling	12	19.4	14	22.6	36	58.1	12.8	0.002*	<0.001**^1 0.13 ^2 0.004**^3
	Photosensitivity	15	27.3	11	20	29	52.7	4.18	0.12	–
	Oral ulcers	4	16.7	5	20.8	15	62.5	2.56	0.28	–
	Discoid lesion	3	17.6	4	23.5	10	58.8	2.14	0.34	–
	Arthritis	23	46	6	12	21	42	17.22	<0.001**	0.03*^1 <0.001**^2 0.58 ^3
	Neurological manifestations	3	25	3	25	6	50	1.47	0.48	–
	Lupus nephritis	9	16.7	7	13	38	70.4	7.51	0.02*	0.26 ^1 0.006*^2 0.36 ^3
	Secondary APLS	3	27.3	2	18.2	6	54.5	0.23	0.9	–
Laboratory findings	CBC profile abnormalities	19	27.1	6	8.6	45	64.3	5.5	0.06	–
	Leukopenia	5	33.9	0	0	7	58.3	2.9	0.2	–
	Anemia	12	22.6	5	9.5	36	67.9	3.96	0.1	–
	Thrombocytopenia	2	40	1	20	2	40	0.8	0.7	–
	C3 level
	Low	15	27.8	8	14.8	31	57.4	0.14	0.93	–
	Normal	13	22.9	6	12.5	29	60.4
	C4 level
	Low	15	27.8	8	14.8	31	57.4	0.14	0.39	–
	Normal	13	22.9	6	12.5	29	60.4
	ANA Pattern
	Homogeneous	11	21.2	3	5.8	38	73
	Speckled	12	33.3	8	22.2	16	44.5	10.9	0.09	–
	Nuclear	2	28.5	2	28.5	3	42.9
	Peri-nuclear	3	42.9	1	14.2	3	42.9
	Anti-dsDNA
	Positive	19	22.9	11	13.3	53	63.9	5.37	0.07	–
	Negative	9	47.4	3	15.8	7	36.8
	ACLA
	Positive	11	35.5	5	16.1	15	48.4	2.06	0.36	–
	Negative	17	23.9	9	12.7	45	63.4
	Anti-dsDNA/LN									0.28 ^1 0.24 ^2 0.02*^3
	Anti-dsDNA+/LN+ (n=58)	15	25.9	11	19	32	55.1	7.76	0.02*
	Anti-dsDNA+/LN- (n=25)	4	16	0	0	21	84
SLEDAI	Mild activity 1–5	18	62.1	2	6.9	9	31			0.002*^1 <0.001**^2 0.30 ^3
	Moderate activity 6–10	8	25.8	6	19.4	17	54.8	33.34	<0.001**
	High activity 11–19	2	6.7	6	20	22	73.3
	Very high activity ≥ 20	0	0	0	0	12	100
	Mean ± SD	8.36±6.67		10±2.08		11.3±6.25		4.21^#	0.03*	0.04*^1 0.03*^2 0.69 ^3
	Range	4 (2–17)		10 (8–12)		10 (3–22)

Notes: Normal WBCs count =4–11 x 10³ /mm³ and leukopenia <4000/ mm³, Normal hemoglobin level=12–16 g/dl and anemia<12g/dl, Normal platelet count=150–400 x 10³ /mm³ and thrombocytopenia <100,000/ mm³, Normal C3 level =90– 210 mg/dl, Normal C4 level =13– 75 mg/dl. χ^2, Chi square test; ^, ANOVA test; ^#Kruskal Wallis test; Post-hoc of ANOVA, Groups with different letters are statistically significant (P<0.05); P1, Del/del versus Del/ins; P2, Ins/ins versus Del/del; P3, Del/ins versus Ins/ins; *Significant; **Highly significant.

Abbreviations: SD, standard deviation; Del, deletion; Ins, insertion; APLS, antiphospholipid syndrome; CBC, complete blood count; C3, complement 3 level; C4, complement 4 level; ANA, antinuclear antibodies; Anti-ds DNA, Anti-double stranded DNA antibodies; ACLA, anticardiolipin antibodies; SLEDAI, systemic lupus erythematosus disease activity index;

Figure 2 TLR9 serum level (ng/L) among the studied groups. A box plot diagram demonstrating a significant difference** (P < 0.001) between the mean serum level of TLR9 in SLE patients (n = 102, mean 397.04 ng/L) and that of controls (n = 102, mean 195.22 ng/L).

Table 4 Relation Between TLR9 Level (ng/L) and Clinical Features, Laboratory Findings and Disease Activity in SLE Patients

Variable		No	TLR9 (ng/L)		t	P
			Mean	SD
Onset	Early (≤20 years)	39	389.05	104.78	0.46	0.65
	Late (>20 years)	63	401.99	155.45
Manifestations	Malar rash N	51	417.95	179.81	1.54	0.13
	Y	51	376.13	71.86
	Hair falling N	40	345.61	53.64	3.16	0.002*
	Y	62	430.22	163.62
	Photosensitivity N	47	367.82	108.93	2.39	0.02*
	Y	55	424.92	130.14
	Oral ulcers N	78	396.26	151.1	0.1	0.92
	Y	24	399.58	83.7
	Discoid lesion N	85	392.91	149.8	0.68	0.5
	Y	17	417.72	40.02
	Arthritis N	52	370.15	99.32	2.04	0.04*
	Y	50	425.01	165.34
	Neurological manifestations N	90	391.86	128.12	1.04	0.3
	Y	12	435.87	199.55
	Lupus nephritis N	48	313.12	65.68	7.06	<0.001**
	Y	54	471.64	142.62
	Secondary APLS N	91	398.09	141.73	0.22	0.83
	Y	11	388.35	105.18
Laboratory findings	CBC Abnormalities N	32	393.57	112.58	1.92	0.06
	Y	70	447.94	140.71
	Leukopenia N	85	397.62	148.03	0.87	0.39
	Y	12	360.02	53.24
	Anemia N	49	432.91	146.13	1.64	0.10
	Y	53	395.38	76
	Thrombocytopenia N	97	404.91	136.81	2.61	0.33
	Y	5	344.4	83.45
Immunological	C3 level
	Low	54	475.09	140.55	7.57	<0.001**
	Normal	48	309.24	60.75
	C4 level
	Low	54	475.09	140.55	7.57	<0.001**
	Normal	48	309.24	60.75
	Anti-dsDNA
	Positive	83	427.95	132.14	5.34	<0.001**
	Negative	19	262.11	58.68
	ACLA
	Positive	31	402.96	215.61	0.29	0.78
	Negative	71	394.46	86.45
	Anti-dsDNA/LN
	Anti-dsDNA+/LN+	58	362.60	85.75	2.21	0.03*
	Anti-dsDNA+/LN-	25	417.08	135.24
SLEDAI	Mild activity 1–5	29	304.83	81.73
	Moderate activity 6–10	31	345.95	31.98	47.09^	<0.001**
	High activity 11–19	30	438.73	114.01
	Very high activity ≥ 20	12	647.65	131.92

Notes: Normal WBCs count =4–11 x 10³ /mm³ and leukopenia <4000/ mm³, Normal hemoglobin level=12–16 g/dl and anemia<12g/dl, Normal platelet count=150–400 x 10³ /mm³ and thrombocytopenia <100,000/ mm³, Normal C3 level =90– 210 mg/dl, Normal C4 level =13– 75 mg/dl. ^ANOVA test; *Significant; **Highly significant.

Abbreviations: SD, standard deviation; Del, deletion; Ins, insertion; APLS, antiphospholipid syndrome; CBC, complete blood count; C3, complement 3 level; C4, complement 4 level; ANA, antinuclear antibodies; Anti-ds DNA, Anti-double stranded DNA antibodies; ACLA, anticardiolipin antibodies; SLEDAI, systemic lupus erythematosus disease activity index; t, independent t-test;

Figure 3 TLR9 level (ng/L) among different HLA-G genotypes in the studied participants. Comparing TLR9 levels among different HLA-G genotypes using Tukey post hoc test demonstrates a significant increase (P < 0.001) in individuals with ins/ins genotype compared to other genotypes whether in SLE patients (n = 102) or control group (n = 102). *Significant, **Highly significant.