Genotypes in matrix metalloproteinase 9 are a risk factor for COPD

Figures & data

Table 1 PCR primers and probes for detecting polymorphisms

Polymorphism	Primers	Probes
MMP-1 G/GG-1607	5′-GTTATGCCACTTAGATGAGGAAATTG (S)	5′-AGATAAGTCATATCCTTTCT
	5′-CATAAACAATACTTCAGTATATCTTGGATTGA (AS)	5′-AGTCATATCTTTCTAATTAT
MMP-2 C/T -1306	5′-CCCTAAAACATTCCCCATA TTCC (S)	5′-CACTCCACCTCTTTA
	5′-GTGACTTCTGAGC TGAGACCTGAA (AS)	5′-AGCACTCTACCTCTT
MMP-9 C/T -1562	5′-GCCTGGCACATAGTAGGCCC (S)	No SphI site (C)
	5′-CTTCCTAGCCAGCCGGCATC (AS)	SphI site 300+260 bp (T)
MMP-9 Gln279Arg	5′-TCTCCCCCTTTCCCACATC (S)	5′-CTCTACACCCGGGACGGCAATG
	5′-TGTGCTGTCTCCGCCTTCT (AS)	5′-ACTCTACACCCAGGACGGCAATGC
MMP-9 CA repeat	5′-6FAM-AAGCCCTTTCTCATGCTGGTGC	Fragment analysis
	5′-TGACAGGCAAGTGCTGACTCAG

Note: The bold, underlined letters are the single nucleotide polymorphisms designed in the probes for allelic discrimination.

Abbreviations: AS, antisense primer; S, sense primer.

Table 2 Summary of demographic and baseline variables plus information on baseline PFT for all participants and those with two visits

	All participants		Participants with 2 visits
	COPDa	Non-COPD	COPDa	Non-COPD
N (%)	123(31.9)	262(68.1)	48 (27.3)	128 (72.7)
Age class (years) N (%)
40–49	6 (4.9)	42 (16.0)	4 (8.3)	15 (11.7)
50–59	30 (24.4)	112 (42.7)	11 (22.9)	54 (42.2)
60–69	56 (45.5)	74 (28.2)	19 (40.0)	41 (32.0)
>=70	31 (25.2)	34 (13.0)	14 (29.2)	18 (14.1)
Ethnicity N (%)
Non-hispanic white	90 (73.2)	162 (61.8)	38 (79.2)	83 (64.8)
Hispanic	20 (16.3)	61 (23.3)	5 (10.4)	28 (21.9)
Other	13 (10.6)	39 (14.9)	5 (10.4)	17 (13.3)
Current smoker yes/no (%)	54/69 (43.9)	109/153 (41.6)	20/28 (41.7)	42/86 (32.8)
Smoking
Duration (years) average (min, max)	40.4 (5.0–60.0)	33.7 (10.0–60.0)	40.2 (6.0–60.0)	33.0 (10.0–60.0)
Smoking, pack-years average (min, max)	60.1 (20.0–220.0)	49.1 (20.0–140.0)	60.0 (24.0–205.0)	51.6 (20.0–140.0)
Baseline %FEV1 (average (min, max))	58.6 (19.0–99.0)	84.1 (40.0–120.0)	61.5 (33.0–87.0)	84.6 (57.0–113.0)

a Using COPD definition I.

Table 3 Distribution of CA repeat alleles

Allele 1	Allele 2	Frequency	Percent
14	14	97	26.5
18	14	1	0.3
19	14	4	1.1
20	14	2	0.5
21	14	74	20.2
21	15	2	0.5
21	19	1	0.3
21	21	39	10.8
22	14	57	15.6
22	15	3	0.8
22	19	2	0.5
22	20	1	0.3
22	21	7	1.9
22	22	17	4.6
23	14	28	7.6
23	15	1	0.3
23	19	1	0.3
23	21	9	2.6
23	22	2	0.5
23	23	6	1.6
24	14	6	1.6
24	21	3	0.8
25	14	1	0.3
26	21	2	0.5

Figure 1 Distribution of CA repeat lengths.

Table 4 Distribution of genotypes in COPD and non-COPD participants (%)

		All	Non-hispanic white			Hispanic
		COPD	Non-COPD	COPD	Non-COPD	COPD	Non-COPD
MMP-9a Gln279Arg	C	35.0	52.1**	34.4	47.8*	40.0	67.2*
	H	46.3	40.2	43.3	42.9	55.0	31.2
	R	18.7	7.7 **	22.2	9.3**	5.0	1.6
	n	123	261	90	161	20	61
MMP-9a C/T-1562	C	68.9	75.9	65.1	73.7	80.0	87.9
	H	26.0	21.7	27.9	23.7	20.0	12.1
	R	5.0	2.4	7.0	2.6	0.0	0.0
	n	119	253	86	156	20	58
MMP-9b CA repeat	SS	24.2	27.6	29.9	36.2	5.0	13.6
	LS	46.7	50.0	42.5	49.0	65.0	54.2
	LL	29.2	22.4	27.6	14.8*	30.0	32.2
	n	120	246	87	149	20	59
MMP-1a	C	26.2	27.2	23.6	19.8	45.0	42.6
	H	46.7	46.3	41.6	49.0	55.0	42.6
	R	27.1	26.5	34.8	31.2	0.0	14.8
	n	122	257	89	157	20	61
a MMP-2	C	62.3	55.0	62.9	53.2	60.0	57.4
	H	32.0	38.0	32.6	40.5	30.0	36.1
	R	5.7	7.0	4.5	6.3	10.0	6.6
	n	122	258	89	158	20	61

a For MMP-9 Gln279Arg, C-1562T, MMP-1, and MMP-2: C=common, H=heterozygote, R=rare.

b For MMP-9 CA repeat length: S=Short, L=Long.

Difference between COPD (by definition I) and non-COPD participants:

** p<0.01,

* p<0.05.

Table 5 Association of genotypes with COPDa

	All	Non-hispanic white	Hispanic
MMP-9Gln279Arg	n	384	251	81
	Heterozyg.	1.8 (1.1, 3.0) *	1.7 (0.9, 3.1)	2.9 (0.9, 9.5)
	Homozyg. rare	3.8 (1.8, 7.9) **	3.5 (1.5, 8.1) *	6.9 (0.4, 137.5)
MMP-9C/T-1562	n	372	242	78
	Heterozyg.	1.3 (0.8, 2.3)	1.4 (0.7, 2.8)	1.7 (0.4, 7.7)
	Homozyg. rare	3.0 (0.8, 10.8)	4.5 (1.1, 10.4) *	NAb
MMP-9c CA repeat	n	366	236	79
	Long/Short	1.1 (0.6, 1.9)	1.2 (0.6, 2.2)	2.4 (0.2, 26.1)
	Long/Long	1.5 (0.8, 3.0)	2.3 (1.0, 5.1) *	2.4 (0.2, 29.0)
MMP-1	n	379	246	81
	Heterozyg.	1.2 (0.7, 2.2)	0.7 (0.3, 1.4)	2.09 (0.61, 7.15)
	Homozyg. rare	1.2 (0.7, 2.4)	0.9 (0.4, 2.0)	NAb
MMP-2	n	380	247	81
	Heterozyg.	0.8 (0.5, 1.4)	0.8 (0.4, 1.5)	0.7 (0.2, 2.4)
	Homozyg. rare	0.7 (0.3, 1.8)	0.6 (0.2, 2.4)	2.3 (0.3, 17.4)

a COPD definition I.

b not applicable due to zero participants with homozygous rare.

c OR (95% CI) relative to homozygous common and Short/Short CA repeats.

* p<0.05,

** p<0.001.

NOTE: Models include an adjustment for current smoking status and linear adjustments for age and smoking duration.

Table 6 MMP-9 C/T-1562, Gln279Arg, and CA repeat haplotype frequencies

Haplotypeb	Relative frequency		Adjusted ORa
	COPDc	Non- COPD	Estimate 95% CI	P value
C-C-S	0.47	0.53	1.0
C-C-L	0.11	0.19	0.8 (0.5–1.2)	0.26
C-R-S	0.01	0.00
C-R-L	0.23	0.15	1.8 (1.1–2.9)	0.02
R-C-S	0.00	0.00
R-C-L	0.00	0.00
R-R-S	0.00	0.03
R-R-L	0.18	0.13	1.7 (1.1–2.7)	0.03

a Logistic model adjustments included age, smoking duration, and current smoking status.

b C-1562T (C=common, R=rare), Exon 6 (C=common, R=rare), CA repeat length (S=Short, L=Long).

c COPD definition I.

Appendix Table 4 Distribution of polymorphisms by COPD status (definition I)

		All participants		Participants with 2 visits
		COPD	Non-COPD	COPD	Non-COPD
MMP-9a Gln279Arg	C	35.0	52.1**	35.4	52.3
	H	46.3	40.2	37.5	41.4
	R	18.7	7.7 **	27.1	6.3
	n	123	261	48	128
MMP-9a C/T-1562	C	68.9	75.9	60.9	75.8
	H	26.0	21.7	32.6	21.8
	R	5.0	2.4	6.5	2.4
	n	119	253	46	124
MMP-9b CA repeat	SS	24.2	27.6	29.8	26.7
	LS	46.7	50.0	34.0	52.5
	LL	29.2	22.4	36.2	20.8
	n	120	246	47	120
MMP-1a	C	26.2	27.2	22.9	25.0
	H	46.7	46.3	47.9	46.9
	R	27.1	26.5	29.2	28.1
	n	122	257	48	128
MMP-2a	C	62.3	55.0	58.3	50.0
	H	32.0	38.0	33.3	42.2
	R	5.7	7.0	8.3	7.8
	n	122	258	48	128

a For MMP-9 Gln279Arg, C-1562T, MMP-1, and MMP-2: C=common, H=heterozygote, R=rare.

b For MMP-9 CA repeat length: S=Short, L=Long.

Difference between COPD (by definition I) and non-COPD participants:

** p<0.01,

* p<0.05.

Appendix Table 5 Association of genotypes with COPD

COPD definition II
		All	Non-hispanic white	Hispanic
Gln279Arg	n	366	238	76
	Heterozyg.	2.04 (1.19, 3.49)*	1.75 (0.92, 3.35)	4.93 (1.26, 19.28)
	Homozyg. rare	3.13 (1.42, 6.94)**	2.77 (1.13, 6.81)*	12.61 (0.58, 275.15)
C/T-1562	n	354	229	73
	Heterozyg.	1.37 (0.77, 2.43)	1.48 (0.74, 2.96)	2.47 (0.55, 11.07)
	Homozyg. rare	2.24 (0.54, 9.30)	3.58 (0.72, 17.69)	NAa
CA repeat	n	348	223	74
	Long-Short	1.35 (0.72, 2.52)	1.23 (0.62, 2.45)	NAa
	Long-Long	2.00 (0.97, 4.12)	1.98 (0.85, 4.63)	NAa
MMP1	n	361	233	76
	Heterozyg.	1.28 (0.69, 2.36)	0.69 (0.32, 1.50)	3.19 (0.78, 12.30)
	Homozyg. rare	1.10 (0.54, 2.24)	0.85 (0.37, 1.93)	NAa
MMP2	n	362	234	76
	Heterozyg.	0.85 (0.50, 1.43)	0.86 (0.46, 1.60)	0.61 (0.15, 2.41)
	Homozyg. rare	0.70 (0.25, 1.99)	0.78 (0.21, 2.90)	1.15 (0.10, 13.96)
COPD definition II
		All	Non-hispanic white	Hispanic
Gln279Arg	n	339	217	74
	Heterozyg.	1.78 (0.98, 3.23)	1.36 (0.65, 2.81)	4.08 (1.02, 16.27)
	Homozyg. rare	3.14 (1.35, 7.30)**	3.32 (1.31, 8.39)*	10.75 (0.50, 230.22)
C/T-1562	n	327	208	71
	Heterozyg.	1.31 (0.68, 2.53)	1.38 (0.63, 3.04)	2.22 (0.45, 10.89)
	Homozyg. rare	3.25 (0.78, 13.62)	5.08 (1.02, 25.18)*	NAb
CA repeat	n	321	202	72
	Long-Short	1.29 (0.64, 2.61)	1.01 (0.46, 2.20)	NAa
	Long-Long	2.08 (0.94, 4.57)	2.16 (0.87, 5.36)	NAa
MMP1	n	334	212	74
	Heterozyg.	1.31 (0.67, 2.54)	0.65 (0.29, 1.49)	3.17 (0.75, 13.41)
	Homozyg. rare	0.75 (0.33, 1.71)	0.55 (0.22, 1.39)	NAa
MMP2	n	335	213	74
	Heterozyg.	0.83 (0.46, 1.51)	0.84 (0.42, 1.70)	0.77 (0.19, 3.12)
	Homozyg. rare	1.03 (0.36, 2.94)	1.16 (0.31, 4.39)	1.38 (0.11, 16.86)

a not applicable due to zero participants with homozygous rare.

b OR (95% CI) relative to homozygous common and Short/Short CA repeats.

* p<0.05,

** p<0.001.

NOTE: Models include an adjustment for current smoking status and linear adjustments for age and smoking duration.