Associations between m-type phospholipase A2 receptor,human leukocyte antigen gene polymorphisms and idiopathic membranous nephropathy

Figures & data

Table 1. Calculation method of eGFR

Gender	Scr (mg/dl)	Formula
Female	≤0.7	eGFR = 144X(Scr%0.7)^−0.329X(0.993)^age
	>0.7	eGFR = 144X(Scr%0.7)-^1.209X(0.993)^age
Male	≤0.9	eGFR = 144X(Scr%0.7)^−0.311X(0.993)^age
	>0.9	eGFR = 144X(Scr%0.7)-^1.209X(0.993)^age

Note: The unit of SCR in the formula is mg/dL.

Table

Experimental instruments	Company
−80°C refrigerator	China’s haier
Piece head (1000 μl)	Invitrogen U.S.
Ependof tube (1 ml)	Eppendorf Germany
TGUIDE M16 Automatic Nucleic Acid Extraction Instrument	Tiangen Biochemical Technology Co., Ltd
QT-1 vortex mixer	Shanghai Qite Analytical Instrument Co., Ltd
Mini-4LC miniature centrifuge	Zhuhai black horse medical instrument co., LTD
TD5A-WS bench top low speed centrifuge	Changsha Xiangyi Centrifuge Instrument Co. Ltd
DK-8D type electric thermostatic water tank	Shanghai Jinghong Experimental Equipment Co., Ltd
Gel imager	Shanghai Peiqing Technology Co., Ltd
FR-110 UV analysis device	Shanghai Furi Technology Co., Ltd
FR – 250 electrophoresis apparatus	Shanghai Furi Technology Co., Ltd
Multi-purpose horizontal electrophoresis tank	Beijing Baijing Biotechnology Co., Ltd
YXQ-LS-30II vertical pressure steam sterilizer	Shanghai Boxun Industrial Co., Ltd. Medical Equipment Factory
2720 Thermal Cycler	ABI
1–10 μl 12 pipettes	discovery
Ultra-clean table	Shanghai EPS Laboratory Equipment Co., Ltd
XW-80A vortex mixer	Shanghai Qite Analytical Instrument Co., Ltd
H1650-W table top micro high speed centrifuge	Changsha Xiangyi Centrifuge Instrument Co. Ltd
DHG-9053A type electric heating constant temperature air blowing drying oven	Shanghai Jinghong Experimental Equipment Co., Ltd
3730xl genetic analyze	ABI
Centrifuge5810R	Eppendorf, Germany
Milli-Q Academic	Millipore
Vc refrigerator BCD – 239	Henan Xinfei Electric Appliance Co. Ltd
HC-TP11-10 medical balance tray	Shanghai Precision Scientific Instrument Co., Ltd
Micro sampler	Eppendorf, Germany

Table

Reagents	Company
Blood genomic DNA extraction kit	Tiangen Biochemical Technology Co., Ltd
Hotstar Taq	Qiagen
primer	Shanghai sangon
PCR reaction buffer	Takara
MgCl2	Takara
dNTP	GENERAY BIOTECH
PCR Marker	NEW ENGLAND Biolabs
agarose	BIOWEST
Ethidium bromide	Shanghai sangon
Bromophenol blue	Shanghai sangon
imLDR Multiplex	Shanghai Tianhao Biological Technology Co., Ltd
SAP	Promega
EXO-I	Epicenter
HI-DI	ABI
GeneScanTM-500	ABI

Table

SNP	Primer
rs2715918F	CACAAGAAAAGAGAGGCTCGATTTGA
rs2715918R	GAAACCCAAGATTCCGTTCTGGT
rs35771982F	GCATGGGAAATGCTGCTGTGTA
rs35771982 R	GAGCACATGAGCAGTAAAACAGTGG
rs3749117F	GCATGGGAAATGCTGCTGTGTA
rs3749117R	GAGCACATGAGCAGTAAAACAGTGG
rs2715928F	CATCCATCCCTGTGTCCACCTC
rs2715928R	TGGTGGTTCCTGCTTGGTTGTT
rs16844715F	CGATATATTAGTGGGGGACCATTTCAC
rs16844715R	GGATGGAGCATCCTGGTGAAAAC
rs4664308F	AGCCCTTCACTCGGAGGATCAC
rs4664308R	GGAGGCTTAATCAGGGGCAGGT
rs2187668F	CAACARTCATTTTACCACATGGTCCTC
rs2187668R	GKTGAAGAACAGGTAATTTGGGTTGATA

Table

SNP	Linking primers
rs2715918RG	TCTCTCGGGTCAATTCGTCCTTTCCGTTCTGGTACCAGTACGGTAAGTAC
rs2715918RA	TGTTCGTGGGCCGGATTAGTTCCGTTCTGGTACCAGTACGGTAAGCAT
rs2715918RP	GACTCTGGCAGCTCTTGAATTTCTAA
rs35771982FG	TTCCGCGTTCGGACTGATATGACCACTGCCAGCCAGCTTG
rs35771982FC	TACGGTTATTCGGGCTCCTGTGACCACTGCCAGCCAGCTTC
rs35771982FP	TTCATCCAGCTGATTGAGGCCTTTTTT
rs3749117FC	TCTCTCGGGTCAATTCGTCCTTCATCCAGCTGATTGAGGCCAAC
rs3749117FT	TGTTCGTGGGCCGGATTAGTCATCCAGCTGATTGAGGCCGAT
rs3749117FP	CCACACCTCCACTGTTTTACTGCTT
rs2715928FG	TCTCTCGGGTCAATTCGTCCTTCTTGAGGGCAGAGCACAGGAAGTG
rs2715928FA	TGTTCGTGGGCCGGATTAGTCTTGAGGGCAGAGCACAGGAAGTA
rs2715928FP	GCTAGCATGCTGTGAAGAATGTAACC
rs16844715RC	TTCCGCGTTCGGACTGATATGGAGCATCCTGGTGAAAACTTGG
rs16844715RT	TACGGTTATTCGGGCTCCTGTGGAGCATCCTGGTGAAAACTCGA
rs16844715RP	CATAGTCATTGCTAAAGGAAATGATCAGGT
rs4664308RG	TCTCTCGGGTCAATTCGTCCTTGGGGCAGGTAGAACAAGACCATTCTGAC
rs4664308RA	TGTTCGTGGGCCGGATTAGTGGGGCAGGTAGAACAAGACCATTCTGAT
rs4664308RP	TCTAAAGTGACCAAAGTCAGAGATGCTC T
rs2187668RC	TTCCGCGTTCGGACTGATATGTGACACAWATGAGGCAGCTGAGAGTCAG
rs2187668RT	TACGGTTATTCGGGCTCCTGTGTGACACAWATGAGGCAGCTGAGAGTCAA
rs2187668RP	TGAGGACCATGTGGTAAAATGAYTGTT

Table 2. Clinical features of IMN group and control group

	IMN (n = 86)	Controls (n = 90)	P-value
Male/female	53/33	35/55	0.04
Age (year)	49 ± 13.21	48 ± 13.21	0.605
SBP (mmHg)	130.79 ± 18.55	123.58 ± 14.64	0.005
DBP (mmHg)	84.44 ± 10.63	77.63 ± 9.41	<0.001
BMI (kg/m^2)	24.60 ± 4.53	23.49 ± 3.10	0.59
Scr (μmol/L)	77.14 ± 43.67	57.59 ± 14.21	<0.001
ALB (g/L)	23.53 ± 5.26	40.30 ± 5.0	<0.001
CHOL (mmol/L)	8.91 ± 3.29	4.01 ± 1.0	<0.001
TG (mmol/L)	3.08 ± 2.08	1.05 ± 0.69	<0.001

Table 3. Hardy-Weinberg balance test

SNP	Major/minor		IMN genotype			HWE P value	Controls genotype			HWE P value
	(A/B)		AA	AB	BB		AA	AB	BB
rs16844715	C/T	49		30	7	0.44	31	44	15	0.93
rs2187668	C/T	75		11	0	0.53	85	4	1	0.003
rs2715918	A/G	3		33	50	0.38	0	22	68	0.19
rs2715928	A/G	63		19	4	0.13	35	43	12	0.83
rs35771982	G/C	69		16	1	0.95	41	40	9	0.87
rs3749117	T/C	69		16	1	0.95	41	40	9	0.87
rs4664308	A/G	66		17	3	0.17	46	36	8	0.80

Table 4. Distribution of SNP locus alleles between IMN group and control group

SNP	Major/minor	Allele frequency in IMN		Allele frequency in Control		OR with 95% CI	Allele P	Adjusted P*
		Major	Minor	Major	Minor
rs16844715	C/T	128 (0.74)	44 (0.26)	106 (0.59)	74 (0.41)	2.03 (1.29–3.19)	0.0020	0.0140
rs2715918	A/G	39 (0.23)	133 (0.77)	22 (0.12)	158 (0.88)	2.11 (1.20–3.70)	0.0096	0.0672
rs2715928	A/G	145 (0.84)	27 (0.16)	113 (0.63)	67 (0.37)	3.18 (1.94–5.24)	5.05E-6	3.54E-5
rs35771982	G/C	154 (0.90)	18 (0.10)	122 (0.68)	58 (0.32)	4.07 (2.34–7.08)	7.08E-7	4.96E-6
rs3749117	T/C	154 (0.90)	18 (0.10)	122 (0.68)	58 (0.32)	4.07 (2.34–7.08)	7.08E-7	4.96E-6
rs4664308	A/G	149 (0.87)	23 (0.13)	128 (0.71)	52 (0.29)	2.63 (1.54–4.49)	0.0004	0.0028

Note: * is corrected by Bonferroni.

Table 5. Distribution of SNP locus genotypes between IMN group and control group

SNP	Major/minor A/B	IMN (n = 86)			Controls (n = 90)			Genotype P	Adjusted P*
		AA (%)	AB (%)	BB (%)	AA (%)	AB (%)	BB (%)
rs16844715	C/T	49 (57.0)	30 (34.9)	7 (8.1)	31 (34.4)	44 (48.9)	15 (16.7)	0.009	0.063
rs2715918	A/G	3 (3.5)	33 (38.4)	50 (58.1)	0 (0)	22 (24.4)	68 (75.6)	0.02	0.14
rs2715928	A/G	63 (73.3)	19 (22.1)	4 (4.6)	35 (38.9)	43 (47.8)	12 (13.3)	2.5E-5	1.75E-4
rs35771982	G/C	69 (80.2)	16 (18.6)	1 (1.2)	41 (45.6)	40 (44.4)	9 (10.0)	7.0E-6	4.9E-6
rs3749117	T/C	69 (80.2)	16 (18.6)	1 (1.2)	41 (45.6)	40 (44.4)	9 (10.0)	7.0E-6	4.9E-6
rs4664308	A/G	66 (76.7)	17 (19.8)	3 (3.5)	46 (51.1)	36 (40.0)	8 (8.9)	0.002	0.014

Note: * is corrected by Bonferroni.

Table 6. Association of SNP loci with IMN susceptibility under different genetic models

Model		OR	95% CI	P	Adjusted P*
rs16844715
Additive	CC/TT	3.39	1.28–8.96	0.0140	0.0980
	CT/TT	1.46	0.53–4.00	0.4605	3.2235
Dominant	(CC+CT)/TT	2.25	0.89–5.74	0.8730	6.1110
Recessive	CC/(CT+TT)	2.52	1.38–4.61	0.0027	0.0189
rs2715918
Additive	AA/GG	–	–	0.0470	0.3290
	AG/GG	2.04	1.07–3.89	0.307	2.1490
Dominant	(AA+AG)/GG	2.23	1.18-.21	0.0140	0.0980
Recessive	AA/(AG+GG)	–	–	0.0739	0.5173
rs2715928
Additive	AA/GG	5.40	1.77–16.50	0.0031	0.0217
	AG/GG	1.33	0.38–4.63	0.6588	4.6116
Dominant	(AA+AG)/GG	3.15	1.02–9.71	0.0452	0.3164
Recessive	AA/(AG+GG)	4.30	2.31–8.03	4.48E-6	3.14E-5
rs35771982
Additive	GG/CC	15.15	2.92–78.48	0.0012	0.0084
	GC/CC	3.6	0.47–27.40	0.2161	1.5127
Dominant	(GG+GC)/CC	9.44	1.66–53.73	0.0114	0.0798
Recessive	GG/(GC+CC)	4.85	5.53–9.31	2.03E-6	1.42E-5
rs3749117
Additive	TT/CC	15.15	2.92–78.48	0.0012	0.0084
	TC/CC	3.6	0.47–27.40	0.2161	1.5127
Dominant	(TT+TC)/CC	9.44	1.66–53.73	0.0114	0.0798
Recessive	TT/(TC+CC)	4.85	5.53–9.31	2.03E-6	1.42E-5
rs4664308
Additive	AA/GG	3.83	1.04–14.08	0.0435	0.3045
	AG/GG	1.26	0.30–5.34	0.7545	5.2815
Dominant	(AA+AG)/GG	2.70	0.72–10.06	0.1390	0.9730
Recessive	AA/(AG+GG)	3.16	1.67–5.97	0.0004	0.0028

Note: * is corrected by Bonferroni.

Figure 1. Linkage disequilibrium analysis of PLA2R gene

Table 7. PLA2R haplotype analysis

Block	Haplotypes	Frequencies	Case ratios	Control ratios	P-value
Block 1	H1-GT	0.784	0.895	0.678	7.08E-7
	H2-CC	0.216	0.105	0.322	7.08E-7
Block 2	H3-CA	0.662	0.738	0.588	0.0030
	H4-TG	0.210	0.128	0.288	0.0002
	H5-TA	0.125	0.128	0.123	0.8776

Figure 2. Combination of high-risk and low-risk genotypes under the best model

Table 8. Establishing GMDR model to analyze the interaction between SNPs of PLA2R

Model	Training bal. acc.	Testing bal. acc.	CV consistency	Sign test (P)
rs355771982	0.6766	0.6514	6/10	10 (0.0010)
rs2715928, rs35771982	0.6998	0.6891	10/10	10 (0.0010)
rs2715928, rs35771982, rs4664308	0.7.64	0.6961	7/10	10 (0.0010)

Table 9. Effects of different genotypes on clinical manifestations of IMN

Clinical parameter	rs16844715			rs2715928			rs35771982
	CT/CC	TT	P	GA/AA	GG	P	GC/GG	CC	P
Male/Female	48/31	5/2	0.58	48/33	4/0	0.10	53/33	1/0	0.42
Age (year)	49.68 ± 13.39	50.14 ± 11.88	0.93	49.67 ± 13.28	50.75 ± 13.53	0.87	49.89 ± 13.19	35	0.27
24 h proteinuria quantification (g)	6.30 ± 3.42	6.71 ± 2.97	0.76	6.39 ± 3.44	5.13 ± 0.90	0.06	6.33 ± 3.39	5.80	0.88
CHOL (mmol/L)	8.83 ± 3.38	9.84 ± 2.17	0.44	8.85 ± 3.36	10.20 ± 1.16	0.43	8.88 ± 3.30	11.93	0.36
TG (mmol/L)	3.14 ± 2.15	2.42 ± 1.03	0.38	3.11 ± 2.12	2.62 ± 1.50	0.65	3.08 ± 2.10	3.16	0.97
ALB (g/L)	23.90 ± 5.21	19.33 ± 4.17	0.03*	23.69 ± 5.22	20.38 ± 5.95	0.22	23.65 ± 5.18	13.5	0.06
Scr (μmol/L)	76.70 ± 44.62	82.26 ± 33.06	0.75	76.74 ± 44.64	85.60 ± 11.49	0.69	76.94 ± 43.88	95.30	0.68
BUN (mmol/L)	5.47 ± 2.04	6.69 ± 7.20	0.67	5.64 ± 2.80	4.19 ± 0.40	0.31	5.58 ± 2.77	4.61	0.73
BMI (kg/m^2)	24.54 ± 4.63	25.37 ± 3.49	0.64	24.71 ± 3.79	22.40 ± 13.72	0.76	24.51 ± 4.48	32.40	0.08
eGFR (ml/min)	98.48 ± 24.71	93.05 ± 24.64	0.58	98.45 ± 25.11	89.50 ± 4.80	0.48	98.14 ± 24.73	89.00	0.71
SBP (mmHg)	131.51 ± 18.66	122.71 ± 16.31	0.23	131.01 ± 18.52	126.25 ± 21.45	0.62	130.96 ± 18.59	116.00	0.43
DBP (mmHg)	84.57 ± 10.92	83.00 ± 7.12	0.71	84.46 ± 10.35	84.00 ± 17.76	0.93	84.39 ± 10.69	89.00	0.67

Note: * is corrected by Bonferroni. P* = 0.36.

Table 10. Effects of different genotypes on clinical manifestations of IMN

Clinical parameter	rs3749117			rs4664308
	TC/TT	CC	P	AG/AA	GG	P
Male/Female	52/33	1/0	0.43	50/33	3/0	0.16
Age (year)	49.89 ± 13.19	35.00	0.27	19.73 ± 13.29	49.33 ± 13.20	0.96
24 h proteinuria quantification (g)	6.34 ± 3.39	5.80	0.88	6.36 ± 3.43	5.56 ± 1.06	0.69
CHOL (mmol/L)	8.88 ± 3.30	11.93	0.36	8.81 ± 3.30	11.74 ± 1.99	0.13
TG (mmol/L)	3.08 ± 2.10	3.16	0.97	3.09 ± 2.12	2.87 ± 1.47	0.86
ALB (g/L)	23.65 ± 5.18	13.50	0.06	23.71 ± 5.20	18.67 ± 5.47	0.10
Scr (μmol/L)	76.94 ± 43.88	95.30	0.68	77.10 ± 44.39	78.67 ± 15.23	0.95
BUN (mmol/L)	5.58 ± 2.77	4.61	0.73	5.62 ± 2.79	4.16 ± 0.48	0.37
BMI (kg/m^2)	24.51 ± 4.48	32.40	0.08	24.50 ± 4.53	27.43 ± 4.37	0.27
eGFR (ml/min)	98.14 ± 24.73	89.00	0.71	97.11 ± 25.01	96.11 ± 8.36	0.89
SBP (mmHg)	130.96 ± 18.59	116.00	0.43	131.27 ± 18.51	117.67 ± 17.56	0.21
DBP (mmHg)	84.39 ± 10.69	89.00	0.67	84.57 ± 10.70	71.00 ± 9.85	0.57