Introduction {#s1}
============

Matrix metalloproteinases (MMPs) are zinc-dependent endopeptidases which play a role in connective tissue remodeling.  Circulating levels of MMPs are associated with many cardiovascular diseases (CVDs), including atherosclerosis, myocardial infarction (MI), and heart failure [@pone.0023609-Galis1], [@pone.0023609-Hlatky1], [@pone.0023609-Hutchinson1].  MMP-9 (gelatinase-B) functions in the degradation of type IV and V collagens, and its levels are raised in individuals with hypertension, acute coronary syndrome, and acute MI [@pone.0023609-Hlatky1], [@pone.0023609-Zhou1], [@pone.0023609-Kai1].  MMP-12, (macrophage metalloelastase) is up-regulated in atherosclerotic lesions and aneurysms and may contribute to the activation of other MMPs, which, in turn, degrade other extracellular matrix proteins [@pone.0023609-Liang1].  A recent study found that MMP-12 production by macrophages plays a role in the transition from fatty acids to fibrous plaques during the progression of atherosclerosis [@pone.0023609-Yamada1].  Given MMPs\' associations and possible causal connections with CVD, these enzymes represent potential drug targets for CVD treatment and prevention.  In fact, amlodipine and lercanidipine have been shown to influence MMP-9 plasma levels and activity [@pone.0023609-Zervoudaki1], [@pone.0023609-Zervoudaki2], [@pone.0023609-Martinez1].  ACE inhibitors may also affect MMP levels [@pone.0023609-Jin1], [@pone.0023609-Reinhardt1], [@pone.0023609-Yamamoto1], [@pone.0023609-Liebetrau1].  Attempts have been made to assess associations of MMP gene variants with disease and risk phenotypes [@pone.0023609-Jormsjo1], [@pone.0023609-Blankenberg1], [@pone.0023609-Su1], [@pone.0023609-Joos1], [@pone.0023609-Han1].  For example, individuals carrying the *MMP9* 279Q allele had higher plasma levels of MMP-9 and higher risk of cardiovascular events than patients homozygous for the 279R allele (*P* = 0.02) [@pone.0023609-Blankenberg1].  The *MMP9* R279Q polymorphism is a glutamine to arginine substitution located in the catalytic domain of MMP-9 [@pone.0023609-Blankenberg1], [@pone.0023609-Hirose1] and plausibly represents a loss-of-function mutation [@pone.0023609-Natividad1].  The R668Q variant of *MMP9* lies within exon 12.  This missense mutation lies in the hemopexin-like domain and probably functions in substrate binding [@pone.0023609-Han1].  The *MMP12* −82A\>G variant, which lies in the gene\'s promoter region, affects transcription factor binding affinity, with the A allele associated with increased promoter activity [@pone.0023609-Jormsjo1], [@pone.0023609-Joos1]; this variant has been shown to affect coronary artery luminal diameter [@pone.0023609-Jormsjo1].  The *MMP12* N122S variant lies within the gene\'s coding region; although this variant has been associated with endpoints such as breast cancer prognosis [@pone.0023609-Shin1] and diabetic nephropathy [@pone.0023609-Kure1], the functional significance of the single-nucleotide polymorphism (SNP) is unknown.  Given these and other MMP genetic associations with cardiovascular phenotypes and known molecular interactions between antihypertensive drugs and some MMPs, MMP genes may modify rates of CVD outcomes differently for different antihypertensive treatments.  We tested whether hypertensive participants in the Genetics of Hypertension Associated Treatment (GenHAT) Study genotyped for *MMP9* variants R279Q and R668Q and for *MMP12* variants N122S and −82A\>G and randomized to either the diuretic chlorthalidone, the calcium channel blocker amlodipine (CCB), or the angiotensin converting enzyme (ACE) inhibitor lisinopril had different outcome rates with regard to six CVD phenotypes. ( The doxazosin treatment arm was not included in the pharmacogenetic analyses.  See below.)  All three of these drugs are known to effectively lower blood pressure and all three are commonly prescribed.  The effect of treatment assignment on CVD outcomes has been previously published [@pone.0023609-ALLHAT1], [@pone.0023609-Antihypertensive1].  We sought to determine whether genotype for these variants interacted with treatment assignment to produce a pharmacogenetic association with CVD outcomes. 

Methods {#s2}
=======

Ethics statement {#s2a}
----------------

Participants recruited during the parent Antihypertensive and Lipid-Lowering Treatment to Prevent Heart Attack Trial (ALLHAT) signed informed consent documents; the GenHAT study was approved by the University of Minnesota Institutional Review Board, the University of Alabama at Birmingham Institutional Review Board for Human Use, and the University of Texas Health Science Center at Houston Committee for the Protection of Human Subjects. 

Study Population {#s2b}
----------------

Study participants were part of the GenHAT study, the primary objective of which was to determine whether variants in hypertension susceptibility genes interact with antihypertensive drugs to modify coronary heart disease risk in hypertensive patients [@pone.0023609-Arnett1].  GenHAT is ancillary to the ALLHAT study, a randomized, double-blind clinical trial examining 42,418 hypertensive patients age 55 or older (47% non-White and 46% female) with one or more risk factors for CVD.  Participants were randomized to one of four ALLHAT treatment arms---chlorthalidone, lisinopril, amlodipine, and doxazosin, in a ratio of 1.7∶1∶1∶1, respectively [@pone.0023609-Arnett1].  Due to early discontinuation of the doxazosin arm of ALLHAT [@pone.0023609-ALLHAT2]---and thus fewer clinical outcomes in genotype and treatment groups---we have not included the doxazosin group in these analyses.  The drug doses were titrated to achieve blood pressures lower than 140/90 mmHg.  Detailed descriptions of both ALLHAT and GenHAT have been published elsewhere [@pone.0023609-Arnett1], [@pone.0023609-Davis1]. 

GenHAT genotyped variants in several hypertension-related genes in the 39,114 ALLHAT participants with available DNA, making the study design a post-hoc subgroup analysis of a randomized clinical trial [@pone.0023609-Lynch1].  Participants were excluded from analysis if they were missing genotype data (n = 489 for *MMP9* R279Q, n = 499 for *MMP9* R668Q, n = 1019 for *MMP12* N122S, n = 577 for *MMP12* −82A\>G).  There were 38,698 participants successfully genotyped for at least one of the polymorphisms included in this analysis. 

Outcomes {#s2c}
--------

The primary outcome for both ALLHAT and GenHAT was coronary heart disease (CHD), defined as fatal CHD or nonfatal myocardial infarction (MI).  Secondary outcomes for this study included all-cause mortality (ACM); stroke; chronic heart failure (CHF); combined CHD outcomes (CCHD, defined as CHD death or non-fatal MI plus revascularization procedures plus hospitalized angina); and combined CVD (CCVD, defined as CHD death or non-fatal MI plus stroke, revascularization procedures, angina \[in hospital or outpatient\], heart failure \[in hospital or outpatient\], stroke, and peripheral arterial disease).  Outcomes were reported by the clinical investigator and supported, when possible, by death certificates and hospitalization records.  For a more detailed description of ALLHAT outcomes, see Davis et al. [ @pone.0023609-Davis1]. 

Genotyping {#s2d}
----------

Anonymized samples containing DNA were collected on FTA paper (Fitzco Inc., Maple Plain, MN) and processed as described by Arnett et al. [ @pone.0023609-Arnett1].  Genotyping was performed using a multiplex PCR and a linear immobilized probe research assay for candidate markers (Roche Molecular Systems, Pleasanton, CA, USA) as described by Burns et al. [ @pone.0023609-Burns1].  GenHAT genotyped two SNPs in *MMP9* (R279Q, rs17576 and R668Q, rs2274756) and two SNPs in *MMP12* (N122S, rs652438 and −82A\>G, rs2276109).  The polymorphisms were genotyped in the context of a larger panel of variants selected by the assay\'s designer as candidates for cardiovascular disease.  Genotyping QC was accomplished by preparing a set of 10 trays of replicate DNA samples (n = 767; 2% of GenHAT cohort) which were genotyped along with the others.  For the four variants reported on here, correspondence between replicate genotype calls ranged from 97.6% to 99.1%. 

Statistical analysis {#s2e}
--------------------

STATA version 9.2 (STATA Corporation, College Station, TX) was used for all analyses.  Hardy-Weinberg equilibrium (HWE) tests were performed using chi-square tests.  To test for baseline differences between treatment groups, we used ANOVA for continuous variables and chi-square tests for categorical variables.  Cox proportional regression was used for testing the main effects of MMP genotypes on the outcomes as well as the genotype-by-treatment interactions (the pharmacogenetic effects), resulting in hazard ratios (HR) and ratios of hazard ratios point estimates, respectively.  To test the main effects of the genotypes on the outcomes, we adjusted for age; gender; ethnicity (Hispanic status); race (5 self-reported categories: White, Black, American Indian/Alaskan native, Asian/Pacific Islander, and "other"); smoking status; diabetes status; aspirin use; and baseline values for body mass index (BMI), systolic and diastolic blood pressures, and HDL and LDL cholesterol.  To test for genotype-by-treatment interactions, we created a genotype x treatment parameter and did three separate comparisons: chlorthalidone versus amlodipine, chlorthalidone versus lisinopril, and lisinopril versus amlodipine.  We tested three genetic models for the pharmacogenetic effects: additive (3 genotype categories with a 2 degrees of freedom \[df\] interaction test), dominant (collapsing minor homozygote group with heterozygote group resulting in 2 genotype groups with a 1 df interaction test), and recessive (collapsing common homozygote group with heterozygote group resulting in 2 genotype groups with a 1 df interaction test). 

The previously published GenHAT design paper outlined six primary, *a priori* hypotheses; however, these hypotheses did not include testing the pharmacogenetic effect of *MMP9* or *MMP12* variants.  Therefore, secondary investigations such as this MMP study are considered exploratory and, as such, are not adjusted for multiple comparisons.  Since we performed multiple statistical tests of the pharmacogenetic effects of *MMP9* and *MMP12* variants, caution must be exercised in pronouncing any findings reported here as statistically significant. 

Results {#s3}
=======

[Table 1](#pone-0023609-t001){ref-type="table"} shows the baseline characteristics of the study subjects (n = 38,698, participants with at least one variant included in these analyses).  There were no significant differences between groups except for HDL cholesterol (*P* = 0.04), which was higher in those randomized to amlodipine versus chlorthalidone or lisinopril. 

10.1371/journal.pone.0023609.t001

###### Baseline characteristics for participants (n = 38,698) by treatment group, mean (SD) unless otherwise noted. 

![]( pone.0023609.t001){#pone-0023609-t001-1}

  Characteristic                                Chlorthalidone    Amlodipine      Lisinopril       Doxazosin     *P*-value[\*](#nt102){ref-type="table-fn"}
  -------------------------------------------- ---------------- --------------- --------------- --------------- --------------------------------------------
  Sample size, n (%) by treatment               13,928 (36.0)    8,243 (21.3)    8,273 (21.4)    8,254 (21.3)   
  Age (y)                                         66.8 (7.7)      66.9 (7.7)      66.8 (7.8)      66.7 (7.7)                        0.74
  Race                                                                                                          
  White, n (col %)                               8,451 (60.7)    4,999 (60.7)    5,025 (60.7)    4,976 (60.3)   
  Black, n (col %)                               4,770 (34.3)    2,859 (34.7)    2,839 (34.3)    2,890 (35.0)   
  American Indian/Alaskan native, n (col %)        26 (0.2)        19 (0.2)        18 (0.2)        10 (0.1)     
  Asian/Pacific Islander, n (col %)               170 (1.2)        97 (1.2)        85 (1.0)        95 (1.2)     
  Other, n (col %)                                511 (3.7)        269 (3.3)       306 (3.7)       283 (3.4)                        0.64
  Hispanic, n (%)                                2,722 (19.5)    1,558 (18.9)    1,639 (19.8)    1,618 (19.6)                       0.86
  Women, n (%)                                   6,552 (47.0)    3,926 (47.6)    3,831 (46.3)    3,817 (46.2)                       0.22
  Previous antihypertensive treatment, n (%)    12,571 (90.3)    7,472 (90.7)    7,454 (90.1)    7,447 (90.2)                       0.66
  Blood pressure at baseline, mm Hg                                                                             
  All participants                                                                                              
   SBP                                           146.2 (15.7)    146.2 (15.7)    146.5 (15.6)    146.3 (15.8)                       0.67
   DBP                                           84.1 (10.0)      83.9 (10.2)     84.1 (10.0)     83.9 (10.0)                       0.41
  Treated at baseline                                                                                           
   SBP                                           145.2 (15.6)    145.1 (15.6)    145.4 (15.5)    145.2 (15.7)                       0.75
   DBP                                           83.5 (10.0)      83.3 (10.1)     83.6 (9.9)      83.4 (9.9)                        0.30
  Untreated at baseline                                                                                         
   SBP                                           156.1 (12.0)    156.6 (12.2)    156.4 (12.3)    156.8 (12.5)                       0.57
   DBP                                            89.5 (9.0)      89.7 (9.5)      89.1 (9.3)      89.4 (9.5)                        0.63
  Eligibility risk factors                                                                                      
  Current cigarette smoker, n (%)                3,061 (22.0)    1,810 (22.0)    1,817 (22.0)    1,795 (21.8)                       0.98
  Type 2 diabetes, n (%)                         4,983 (35.8)    3,008 (36.5)    2,899 (35.0)    2,891 (35.0)                       0.15
  HDL cholesterol \<35 mg/dL, n (%)              1,666 (12.0)     939 (11.4)      976 (11.8)      973 (11.8)                        0.65
  LVH by electrocardiogram, n (%)                2,255 (16.2)    1,398 (17.0)    1,342 (16.2)    1,354 (16.4)                       0.47
  Body mass index, kg/m^2^                        29.7 (6.1)      29.8 (6.3)      29.8 (6.2)      29.7 (5.9)                        0.32
  Fasting glucose, mg/dL                         123.4 (58.6)    123.1 (57.5)    122.4 (55.6)    121.9 (55.9)                       0.35
  LDL cholesterol, mg/dL                         136.0 (37.3)    135.7 (37.3)    135.8 (36.4)    135.2 (36.3)                       0.60
  HDL cholesterol, mg/dL                         46.8 (14.9)      47.2 (14.7)     46.6 (14.6)     46.6 (14.4)                     **0.04**
  Fasting triglycerides, mg/dL                  176.7 (130.9)    176.6 (132.8)   175.5 (138.5)   174.1 (137.6)                      0.53
  Aspirin use, n (%)                             4,986 (35.8)    3,002 (36.4)    3,021 (36.5)    3,026 (36.7)                       0.54
  *MMP9* R279Q (A\>G) (rs17576)                                                                                 
  AA                                             5,955 (42.8)    3,422 (41.6)    3,596 (43.6)    3,550 (43.1)   
  AG                                             6,257 (45.0)    3,783 (46.0)    3,633 (44.0)    3,675 (44.6)   
  GG                                             1,691 (12.2)    1,026 (12.5)    1,024 (12.4)    1,013 (12.3)                       0.21
  *MMP9* R668Q (G\>A) (rs2274756)                                                                               
  GG                                            10,004 (72.0)    5,827 (70.8)    5,916 (71.7)    5,939 (72.1)   
  GA                                             3,564 (25.7)    2,196 (26.7)    2,121 (25.7)    2,085 (25.3)   
  AA                                              326 (2.4)        207 (2.5)       219 (2.7)       211 (2.6)                        0.33
  *MMP12* −82A\>G (rs2276109)                                                                                   
  AA                                            11,648 (84.0)    6,899 (84.0)    6,903 (83.8)    6,926 (84.3)   
  AG                                             2,083 (15.0)    1,242 (15.1)    1,269 (15.4)    1,216 (14.8)   
  GG                                              137 (1.0)        68 (0.8)        68 (0.8)        78 (1.0)                         0.74
  *MMP12* N122S (A\>G) (rs652438)                                                                               
  AA                                            11,029 (80.6)    6,562 (80.8)    6,652 (81.5)    6,565 (80.8)   
  AG                                             2,423 (17.7)    1,415 (17.4)    1,400 (17.2)    1,426 (17.6)   
  GG                                              235 (1.7)        145 (1.8)       111 (1.4)       132 (1.6)                        0.31

SBP = systolic blood pressure, DBP = diastolic blood pressure, LVH = left ventricular hypertrophy. 

\*test of differences between genotype groups: ANOVA for continuous variables, chi-square for categorical variables. 

The *MMP9* R279Q and *MMP12* −82A\>G genotype frequencies were in Hardy-Weinberg equilibrium (HWE) when tested within race/ethnicity groups.  The *MMP9* R668Q genotype frequencies were in HWE equilibrium for the Black, American Indian/Alaskan native, Asian/Pacific islander, and "other" groups, but not for the White group (*P* = 0.04).  After stratifying Whites by Hispanic status, the *MMP9* R668Q genotype frequencies were in HWE for non-Hispanic Whites, but not for Hispanic Whites (*P* = 0.001).  The *MMP12* N122S genotype frequencies were in HWE for the Asian/Pacific islander and "other" groups, but not for the Black or White groups.  The American Indian/Alaskan native group included no minor *MMP12* N122S homozygotes (ie, GG).  After stratifying Whites and Blacks by Hispanic status, *MMP12* N122S genotype frequencies were in HWE for all groups except Black non-Hispanics (*P* = 0.0003). 

Main effects of *MMP9* and *MMP12* variants on clinical outcomes {#s3a}
----------------------------------------------------------------

The effects of ALLHAT treatment assignments on CVD outcomes have been published elsewhere [@pone.0023609-ALLHAT1], [@pone.0023609-Antihypertensive1], [@pone.0023609-ALLHAT2].  Hazard ratios for main effects of genetic variants on ALLHAT clinical outcomes in GenHAT are shown in [Table 2](#pone-0023609-t002){ref-type="table"}.  After adjusting for baseline characteristics, the association between the *MMP9* R279Q variant and stroke was modestly significant, with participants carrying the minor G allele having decreased risk of stroke (HR = 1.00 for AA, 0.87 for GA, 0.91 for GG; *P* = 0.04, additive genetic model).  The associations between the *MMP12* N122S variant and CHD and CCHD were also modestly significant (*P* = 0.04 and *P* = 0.01, respectively), with participants with the minor allele homozygote (GG) genotype having a 30% increased risk of CHD or CCHD compared to participants having the common AA genotype (for CHD HR = 1.00 for AA, 0.95 for AG, 1.36 for GG; *P* = 0.04; for CCHD HR = 1.00 for AA, 0.95 for AG, 1.31 for GG; *P* = 0.01).  The effects of the *MMP9* R668Q and *MMP12* −82A\>G variants were not significant for any outcome. 

10.1371/journal.pone.0023609.t002

###### Main effects of MMP genetic variants on clinical outcomes, all treatments combined. 

![]( pone.0023609.t002){#pone-0023609-t002-2}

                                 Number of events (event rate per 1000 p-y)   Adjusted[\*](#nt104){ref-type="table-fn"} Hazard Ratios (95% CI)                                                                                                                 
  ----------------------------- -------------------------------------------- ------------------------------------------------------------------ -------- -------- -------- -------- -------------- -------------- -------------- -------------- -------------- --------------
  *MMP9* R279Q (rs17576)                                                                                                                                                                                                                                       
  AA                                                1373                                                    2573                                  4368     2268     777      1149        1.00           1.00           1.00           1.00           1.00           1.00
                                                   (19.0)                                                  (37.3)                                (68.1)   (29.0)   (10.6)   (15.9)                                                                             
  GA                                                1478                                                    2738                                  4529     2319     731      1158        1.00           1.01           0.97           0.96           0.87           0.92
                                                   (19.5)                                                  (37.8)                                (67.2)   (28.1)   (9.5)    (15.3)   (0.93--1.08)   (0.96--1.07)   (0.93--1.02)   (0.90--1.02)   (0.79--0.97)   (0.85--1.00)
  GG                                                420                                                     767                                   1275     618      202      347         1.00           1.01           0.99           0.93           0.91           1.02
                                                   (20.3)                                                  (38.9)                                (69.3)   (27.4)   (9.6)    (16.8)   (0.90--1.14)   (0.93--1.10)   (0.93--1.06)   (0.85--1.02)   (0.78--1.07)   (0.90--1.16)
  *P*                                                                                                                                                                                    0.99           0.92           0.51           0.20         **0.04**         0.10
  *MMP9* R668Q (rs2274756)                                                                                                                                                                                                                                     
  GG                                                2360                                                    4376                                  7311     3738     1240     1897        1.00           1.00           1.00           1.00           1.00           1.00
                                                   (19.6)                                                  (38.0)                                (68.3)   (28.5)   (10.2)   (15.7)                                                                             
  GA                                                817                                                     1538                                  2579     1323     425      688         0.95           0.97           0.97           0.97           0.94           0.99
                                                   (18.6)                                                  (36.6)                                (66.1)   (27.7)   (9.5)    (15.7)   (0.87--1.03)   (0.92--1.04)   (0.93--1.02)   (0.91--1.03)   (0.84--1.06)   (0.90--1.09)
  AA                                                 94                                                     164                                   275      133       45       67         1.15           1.10           1.06           0.96           0.99           1.00
                                                   (22.4)                                                  (41.0)                                (74.0)   (29.3)   (10.5)   (16.0)   (0.93--1.43)   (0.93--1.29)   (0.94--1.21)   (0.80--1.15)   (0.72--1.35)   (0.77--1.29)
  *P*                                                                                                                                                                                    0.16           0.34           0.31           0.59           0.61           0.98
  *MMP12* −82A\>G (rs2276109)                                                                                                                                                                                                                                  
  AA                                                2692                                                    4991                                  8409     4409     1445     2161        1.00           1.00           1.00           1.00           1.00           1.00
                                                   (19.1)                                                  (37.0)                                (67.0)   (28.7)   (10.1)   (15.3)                                                                             
  AG                                                535                                                     1008                                  1624     736      245      449         0.97           0.96           0.96           0.93           1.00           1.05
                                                   (20.8)                                                  (41.4)                                (71.9)   (26.5)   (9.4)    (17.5)   (0.88--1.08)   (0.90--1.04)   (0.91--1.02)   (0.85--1.01)   (0.86--1.15)   (0.94--1.18)
  GG                                                 35                                                      67                                   116       47       19       32         0.99           1.00           1.12           0.97           1.32           1.26
                                                   (22.4)                                                  (45.2)                                (87.3)   (27.9)   (12.0)   (20.6)   (0.68--1.42)   (0.77--1.29)   (0.92--1.36)   (0.71--1.32)   (0.81--2.13)   (0.86--1.83)
  *P*                                                                                                                                                                                    0.87           0.63           0.24           0.20           0.53           0.36
  *MMP12* N122S (rs652438)                                                                                                                                                                                                                                     
  AA                                                2651                                                    4969                                  8210     4143     1334     2126        1.00           1.00           1.00           1.00           1.00           1.00
                                                   (19.7)                                                  (38.7)                                (68.8)   (28.4)   (9.8)    (15.8)                                                                             
  AG                                                503                                                     911                                   1621     893      321      432         0.95           0.95           0.96           0.98           1.10           0.97
                                                   (17.2)                                                  (32.4)                                (61.9)   (28.2)   (10.9)   (14.9)   (0.86--1.05)   (0.88--1.02)   (0.91--1.02)   (0.91--1.06)   (0.96--1.25)   (0.87--1.09)
  GG                                                 60                                                     102                                   169       82       29       45         1.36           1.31           1.14           0.95           1.01           1.13
                                                   (22.5)                                                  (40.1)                                (71.2)   (27.3)   (10.7)   (16.7)   (1.04--1.79)   (1.06--1.62)   (0.96--1.34)   (0.76--1.20)   (0.68--1.49)   (0.83--1.55)
  *P*                                                                                                                                                                                  **0.04**       **0.01**         0.12           0.83           0.39           0.64

CHD, coronary heart disease defined as fatal CHD or nonfatal myocardial infarction; CCHD, combined CHD defined as CHD death or non-fatal myocardial infarction plus revascularization procedures plus hospitalized angina; CCVD, combined cardiovascular disease defined as CHD death or non-fatal MI plus stroke, revascularization procedures, angina, heart failure, stroke, and peripheral arterial disease; ACM, all-cause mortality; CHF, .chronic heart failure. 

\*adjusted for age, sex, race, Hispanic status, smoking status, diabetes status, aspirin use, and baseline values for BMI, SBP, DBP, HDL- and LDL-cholesterol. 

Pharmacogenetic effects of *MMP9* and *MMP12* variants on clinical outcomes {#s3b}
---------------------------------------------------------------------------

[Tables 3](#pone-0023609-t003){ref-type="table"}, [4](#pone-0023609-t004){ref-type="table"}, [5](#pone-0023609-t005){ref-type="table"}, [6](#pone-0023609-t006){ref-type="table"}, [7](#pone-0023609-t007){ref-type="table"}, [8](#pone-0023609-t008){ref-type="table"} show ALLHAT clinical outcome frequencies and rates by genotype and treatment group for CHD, CCHD, CCVD, ACM, stroke, and CHF, respectively.  Also shown are genotype-specific treatment effects and gene-by-treatment interaction *P*-values for chlorthalidone versus amlodipine, chlorthalidone versus lisinopril, and lisinopril versus amlodipine.  Treatment effects differed by genotype for several outcomes. 

10.1371/journal.pone.0023609.t003

###### Pharmacogenetic effects of MMP genetic variants on CHD. 

![]( pone.0023609.t003){#pone-0023609-t003-3}

                                 Number of events (Event rate per 1000 p-y)   Genotype-specific treatment effects (Hazard Ratio (95% CI))   Pharmacogenetic effects[\*](#nt106){ref-type="table-fn"}                                                                                          
  ----------------------------- -------------------------------------------- ------------------------------------------------------------- ---------------------------------------------------------- -------------- -------------- -------------- ---------------- ------------------------- -------------------------
  *MMP9* R279Q (rs17576)                                                                                                                                                                                                                                                                      
  AA                                                544                                                   285                                                         311                                  1.12           1.06           1.06       *P* ~A~ = 0.11       *P* ~A~ = 0.18            *P* ~A~ = 0.56
                                                   (20.0)                                               (17.9)                                                       (18.9)                            (0.97--1.29)   (0.92--1.21)   (0.90--1.24)                                             
  AG                                                550                                                   343                                                         307                                  0.97           1.03           0.94       *P* ~D~ = 0.07       *P* ~D~ = 0.39            *P* ~D~ = 0.38
                                                   (19.0)                                               (19.7)                                                       (18.5)                            (0.84--1.11)   (0.89--1.18)   (0.81--1.10)                                             
  GG                                                137                                                   100                                                         101                                  0.84           0.81           1.04       *P* ~R~ = 0.13       *P* ~R~ = 0.07            *P* ~R~ = 0.78
                                                   (17.7)                                               (21.1)                                                       (22.0)                            (0.65--1.08)   (0.62--1.04)   (0.79--1.37)                                             
  *MMP9* R668Q (rs2274756)                                                                                                                                                                                                                                                                    
  GG                                                902                                                   523                                                         517                                  1.01           1.02           0.98       *P* ~A~ = 0.13       *P* ~A~ = 0.45            *P* ~A~ = 0.61
                                                   (19.7)                                               (19.5)                                                       (19.2)                            (0.91--1.12)   (0.92--1.14)   (0.87--1.11)                                             
  GA                                                303                                                   177                                                         180                                  1.07           0.99           1.08       *P* ~D~ = 0.97       *P* ~D~ = 0.53            *P* ~D~ = 0.60
                                                   (18.3)                                               (17.2)                                                       (18.6)                            (0.89--1.29)   (0.82--1.19)   (0.88--1.33)                                             
  AA                                                 25                                                   27                                                           24                                  0.59           0.74           0.84       *P* ~R~ = 0.05       *P* ~R~ = 0.22            *P* ~R~ = 0.50
                                                   (16.9)                                               (28.5)                                                       (23.8)                            (0.34--1.02)   (0.42--1.30)   (0.48--1.46)                                             
  *MMP12* −82A\>G (rs2276109)                                                                                                                                                                                                                                                                 
  AA                                                1006                                                  610                                                         591                                  0.98           1.00           0.98       *P* ~A~ = 0.52       *P* ~A~ = 0.19            *P* ~A~ = 0.13
                                                   (18.8)                                               (19.2)                                                       (18.8)                            (0.88--1.08)   (0.90--1.10)   (0.88--1.10)                                             
  AG                                                200                                                   108                                                         125                                  1.14           0.98           1.16       *P* ~D~ = 0.25       *P* ~D~ = 0.77            *P* ~D~ = 0.44
                                                   (20.9)                                               (18.4)                                                       (21.3)                            (0.90--1.44)   (0.79--1.23)   (0.89--1.50)                                             
  GG                                                 18                                                    9                                                           3                                   1.00           3.25           0.33       *P* ~R~ = 0.96       *P* ~R~ = 0.07            *P* ~R~ = 0.10
                                                   (28.9)                                               (28.3)                                                       (9.3)                             (0.45--2.22)   (0.96--11.0)   (0.09--1.22)                                             
  *MMP12* N122S (rs652438)                                                                                                                                                                                                                                                                    
  AA                                                1003                                                  593                                                         563                                  1.01           1.07           0.94       *P* ~A~ = 0.91   ***P*** **~A~ = 0.005**   ***P*** **~A~ = 0.041**
                                                   (19.8)                                               (19.6)                                                       (18.5)                            (0.91--1.12)   (0.97--1.19)   (0.84--1.06)                                             
  AG                                                182                                                   113                                                         128                                  0.96           0.80           1.19       *P* ~D~ = 0.66   ***P*** **~D~ = 0.005**   ***P*** **~D~ = 0.037**
                                                   (16.3)                                               (17.0)                                                       (20.3)                            (0.76--1.21)   (0.64--1.01)   (0.93--1.54)                                             
  GG                                                 21                                                   13                                                           19                                  0.97           0.49           1.93       *P* ~R~ = 0.89   ***P*** **~R~ = 0.019**  
                                                   (19.7)                                               (20.6)                                                       (40.9)                            (0.48--1.93)   (0.26--0.91)   (0.95--3.92)                                             

*P* ~A~ = *P*-value for additive genetic model, *P* ~D~ = *P*-value for dominant genetic model, *P* ~R~ = *P*-value for recessive genetic model. 

\**P*-value for gene-by-treatment interaction. 

CHD, coronary heart disease defined as fatal CHD or nonfatal myocardial infarction; CHL, chlorthalidone treatment group; AML, amlodipine treatment group; LIS, lisinopril treatment group. 

10.1371/journal.pone.0023609.t004

###### Pharmacogenetic effects of MMP genetic variants on CCHD. 

![]( pone.0023609.t004){#pone-0023609-t004-4}

                                 Number of events (event rate per 1000 p-y)   Genotype-specific treatment effects: Hazard Ratio (95% CI)   Pharmacogenetic effects[\*](#nt109){ref-type="table-fn"}                                                                                                   
  ----------------------------- -------------------------------------------- ------------------------------------------------------------ ---------------------------------------------------------- -------------- -------------- -------------- ------------------------- ------------------------- ----------------
  *MMP9* R279Q (rs17576)                                                                                                                                                                                                                                                                              
  AA                                                952                                                  556                                                         569                                  1.00           1.01           0.99           *P* ~A~ = 0.22            *P* ~A~ = 0.12        *P* ~A~ = 0.71
                                                   (36.5)                                               (36.6)                                                      (36.3)                            (0.90--1.11)   (0.91--1.12)   (0.88--1.11)                                                      
  AG                                                1022                                                 602                                                         599                                  1.03           0.97           1.06           *P* ~D~ = 0.91            *P* ~D~ = 0.29        *P* ~D~ = 0.40
                                                   (37.2)                                               (36.1)                                                      (38.1)                            (0.93--1.14)   (0.88--1.08)   (0.94--1.18)                                                      
  GG                                                256                                                  182                                                         186                                  0.85           0.81           1.06           *P* ~R~ = 0.09        ***P*** **~R~ = 0.045**   *P* ~R~ = 0.78
                                                   (34.5)                                               (40.6)                                                      (42.9)                            (0.70--1.03)   (0.67--0.97)   (0.86--1.30)                                                      
  *MMP9* R668Q (rs2274756)                                                                                                                                                                                                                                                                            
  GG                                                1615                                                 946                                                         970                                  1.00           0.97           1.03           *P* ~A~ = 0.12            *P* ~A~ = 0.30        *P* ~A~ = 0.84
                                                   (36.9)                                               (36.9)                                                      (37.9)                            (0.92--1.08)   (0.90--1.05)   (0.94--1.12)                                                      
  GA                                                565                                                  350                                                         346                                  1.01           0.96           1.05           *P* ~D~ = 0.65            *P* ~D~ = 0.54        *P* ~D~ = 0.89
                                                   (35.9)                                               (35.7)                                                      (37.6)                            (0.88--1.15)   (0.84--1.09)   (0.91--1.22)                                                      
  AA                                                 45                                                   44                                                          43                                  0.64           0.71           0.93       ***P*** **~R~ = 0.038**       *P* ~R~ = 0.13        *P* ~R~ = 0.60
                                                   (31.6)                                               (49.2)                                                      (45.4)                            (0.42--0.97)   (0.47--1.08)   (0.61--1.42)                                                      
  *MMP12* −82A\>G (rs2276109)                                                                                                                                                                                                                                                                         
  AA                                                1817                                                 1109                                                        1120                                 0.97           0.95           1.03           *P* ~A~ = 0.48            *P* ~A~ = 0.18        *P* ~A~ = 0.76
                                                   (35.5)                                               (36.5)                                                      (37.6)                            (0.90--1.05)   (0.88--1.02)   (0.95--1.12)                                                      
  AG                                                370                                                  218                                                         226                                  1.04           1.00           1.04           *P* ~D~ = 0.35            *P* ~D~ = 0.31        *P* ~D~ = 0.95
                                                   (40.8)                                               (39.3)                                                      (40.8)                            (0.88--1.23)   (0.85--1.18)   (0.86--1.25)                                                      
  GG                                                 32                                                   12                                                          9                                   1.35           1.91           0.72           *P* ~R~ = 0.33            *P* ~R~ = 0.08        *P* ~R~ = 0.46
                                                   (54.1)                                               (39.4)                                                      (29.1)                            (0.70--2.63)   (0.91--4.01)   (0.30--1.71)                                                      
  *MMP12* N122S (rs652438)                                                                                                                                                                                                                                                                            
  AA                                                1831                                                 1092                                                        1092                                 1.00           1.00           1.00           *P* ~A~ = 0.45        ***P*** **~A~ = 0.023**   *P* ~A~ = 0.14
                                                   (38.0)                                               (37.8)                                                      (37.8)                            (0.93--1.08)   (0.93--1.08)   (0.92--1.09)                                                      
  AG                                                319                                                  209                                                         227                                  0.90           0.79           1.15           *P* ~D~ = 0.39        ***P*** **~D~ = 0.008**   *P* ~D~ = 0.10
                                                   (29.7)                                               (32.9)                                                      (37.7)                            (0.76--1.07)   (0.66--0.93)   (0.95--1.38)                                                      
  GG                                                 40                                                   20                                                          24                                  1.18           0.74           1.58           *P* ~R~ = 0.52            *P* ~R~ = 0.29        *P* ~R~ = 0.14
                                                   (39.1)                                               (33.1)                                                      (53.5)                            (0.69--2.01)   (0.45--1.23)   (0.87--2.85)                                                      

*P* ~A~ = *P*-value for additive genetic model, *P* ~D~ = *P*-value for dominant genetic model, *P* ~R~ = *P*-value for recessive genetic model. 

\**P*-value for gene-by-treatment interaction. 

CCHD, combined CHD defined as CHD death or non-fatal myocardial infarction plus revascularization procedures plus hospitalized angina; CHL, chlorthalidone treatment group; AML, amlodipine treatment group; LIS, lisinopril treatment group. 

10.1371/journal.pone.0023609.t005

###### Pharmacogenetic effects of MMP genetic variants on CCVD. 

![]( pone.0023609.t005){#pone-0023609-t005-5}

                                 Number of events (event rate per 1000 p-y)   Genotype-specific treatment effects: Hazard Ratio (95% CI)   Pharmacogenetic effects[\*](#nt112){ref-type="table-fn"}                                                                                                   
  ----------------------------- -------------------------------------------- ------------------------------------------------------------ ---------------------------------------------------------- -------------- -------------- -------------- ------------------------- ------------------------- ----------------
  *MMP9* R279Q (rs17576)                                                                                                                                                                                                                                                                              
  AA                                                1552                                                 952                                                         963                                  0.94           0.96           0.98       ***P*** **~A~ = 0.038**   ***P*** **~A~ = 0.05**    *P* ~A~ = 0.15
                                                   (63.5)                                               (67.6)                                                      (66.2)                            (0.87--1.02)   (0.89--1.04)   (0.90--1.07)                                                      
  AG                                                1631                                                 978                                                         1012                                 1.01           0.91           1.11           *P* ~D~ = 0.67            *P* ~D~ = 0.10        *P* ~D~ = 0.06
                                                   (63.4)                                               (62.9)                                                      (70.0)                            (0.93--1.09)   (0.84--0.98)   (1.02--1.21)                                                      
  GG                                                413                                                  301                                                         303                                  0.81           0.78           1.04       ***P*** **~R~ = 0.024**   ***P*** **~R~ = 0.026**   *P* ~R~ = 0.98
                                                   (58.8)                                               (72.5)                                                      (75.7)                            (0.70--0.94)   (0.67--0.90)   (0.89--1.22)                                                      
  *MMP9* R668Q (rs2274756)                                                                                                                                                                                                                                                                            
  GG                                                2602                                                 1600                                                        1617                                 0.94           0.93           1.01           *P* ~A~ = 0.36            *P* ~A~ = 0.33        *P* ~A~ = 0.16
                                                   (63.4)                                               (67.3)                                                      (68.1)                            (0.89--1.00)   (0.88--0.99)   (0.94--1.08)                                                      
  GA                                                907                                                  562                                                         597                                  1.00           0.87           1.15           *P* ~D~ = 0.52            *P* ~D~ = 0.17        *P* ~D~ = 0.07
                                                   (61.4)                                               (61.3)                                                      (70.9)                            (0.90--1.11)   (0.78--0.96)   (1.03--1.29)                                                      
  AA                                                 82                                                   65                                                          69                                  0.80           0.78           1.02           *P* ~R~ = 0.28            *P* ~R~ = 0.35        *P* ~R~ = 0.88
                                                   (61.9)                                               (77.4)                                                      (78.9)                            (0.58--1.11)   (0.56--1.07)   (0.73--1.44)                                                      
  *MMP12* −82A\>G (rs2276109)                                                                                                                                                                                                                                                                         
  AA                                                2942                                                 1857                                                        1890                                 0.93           0.90           1.04           *P* ~A~ = 0.21            *P* ~A~ = 0.06        *P* ~A~ = 0.35
                                                   (61.3)                                               (65.7)                                                      (68.6)                            (0.88--0.99)   (0.84--0.95)   (0.98--1.11)                                                      
  AG                                                588                                                  343                                                         371                                  1.05           0.95           1.10           *P* ~D~ = 0.08            *P* ~D~ = 0.16        *P* ~D~ = 0.74
                                                   (69.4)                                               (66.2)                                                      (72.8)                            (0.92--1.20)   (0.84--1.09)   (0.95--1.27)                                                      
  GG                                                 53                                                   23                                                          17                                  1.16           1.75           0.70           *P* ~R~ = 0.41        ***P*** **~R~ = 0.025**   *P* ~R~ = 0.19
                                                  (100.8)                                               (85.9)                                                      (59.0)                            (0.71--1.90)   (1.01--3.02)   (0.37--1.31)                                                      
  *MMP12* N122S (rs652438)                                                                                                                                                                                                                                                                            
  AA                                                2903                                                 1800                                                        1822                                 0.96           0.94           1.02           *P* ~A~ = 0.52            *P* ~A~ = 0.06        *P* ~A~ = 0.19
                                                   (64.2)                                               (67.1)                                                      (68.3)                            (0.90--1.01)   (0.89--1.00)   (0.95--1.09)                                                      
  AG                                                566                                                  350                                                         388                                  0.96           0.81           1.18           *P* ~D~ = 0.76        ***P*** **~D~ = 0.024**   *P* ~D~ = 0.08
                                                   (56.3)                                               (58.8)                                                      (69.5)                            (0.84--1.09)   (0.71--0.92)   (1.02--1.36)                                                      
  GG                                                 59                                                   44                                                          37                                  0.76           0.70           1.10           *P* ~R~ = 0.25            *P* ~R~ = 0.19        *P* ~R~ = 0.83
                                                   (60.9)                                               (80.2)                                                      (88.6)                            (0.52--1.13)   (0.46--1.05)   (0.71--1.71)                                                      

*P* ~A~ = *P*-value for additive genetic model, *P* ~D~ = *P*-value for dominant genetic model, *P* ~R~ = *P*-value for recessive genetic model. 

\**P*-value for gene-by-treatment interaction. 

CCVD, combined cardiovascular disease defined as CHD death or non-fatal MI plus stroke, revascularization procedures, angina, heart failure, stroke, and peripheral arterial disease; CHL, chlorthalidone treatment group; AML, amlodipine treatment group; LIS, lisinopril treatment group. 

10.1371/journal.pone.0023609.t006

###### Pharmacogenetic effects of MMP genetic variants on ACM. 

![]( pone.0023609.t006){#pone-0023609-t006-6}

                                 Number of events (event rate per 1000 p-y)   Genotype-specific treatment effects: Hazard Ratio (95% CI)   Pharmacogenetic effects[\*](#nt115){ref-type="table-fn"}                                                                                          
  ----------------------------- -------------------------------------------- ------------------------------------------------------------ ---------------------------------------------------------- -------------- -------------- -------------- ------------------------- ---------------- -------------------------
  *MMP9* R279Q (rs17576)                                                                                                                                                                                                                                                                     
  AA                                                858                                                  465                                                         519                                  1.08           1.00           1.07           *P* ~A~ = 0.72        *P* ~A~ = 0.87       *P* ~A~ = 0.76
                                                   (29.6)                                               (27.5)                                                      (29.5)                            (0.96--1.21)   (0.90--1.12)   (0.95--1.22)                                             
  AG                                                883                                                  510                                                         526                                  1.04           0.97           1.08           *P* ~D~ = 0.53        *P* ~D~ = 0.67       *P* ~D~ = 0.85
                                                   (28.6)                                               (27.5)                                                      (29.6)                            (0.93--1.16)   (0.87--1.08)   (0.96--1.22)                                             
  GG                                                229                                                  141                                                         138                                  0.98           1.00           0.98           *P* ~R~ = 0.50        *P* ~R~ = 0.87       *P* ~R~ = 0.46
                                                   (27.6)                                               (28.0)                                                      (27.6)                            (0.79--1.21)   (0.81--1.24)   (0.77--1.24)                                             
  *MMP9* R668Q (rs2274756)                                                                                                                                                                                                                                                                   
  GG                                                1420                                                 790                                                         859                                  1.05           0.97           1.08           *P* ~A~ = 0.97        *P* ~A~ = 0.83       *P* ~A~ = 0.81
                                                   (29.1)                                               (27.6)                                                      (29.8)                            (0.96--1.15)   (0.89--1.06)   (0.98--1.19)                                             
  GA                                                501                                                  294                                                         291                                  1.05           1.02           1.03           *P* ~D~ = 0.94        *P* ~D~ = 0.54       *P* ~D~ = 0.55
                                                   (28.4)                                               (27.0)                                                      (27.8)                            (0.91--1.21)   (0.89--1.18)   (0.87--1.21)                                             
  AA                                                 47                                                   30                                                          32                                  1.00           1.06           0.96           *P* ~R~ = 0.81        *P* ~R~ = 0.87       *P* ~R~ = 0.71
                                                   (30.0)                                               (30.1)                                                      (29.7)                            (0.63--1.57)   (0.67--1.66)   (0.58--1.58)                                             
  *MMP12* −82A\>G (rs2276109)                                                                                                                                                                                                                                                                
  AA                                                1646                                                 951                                                         1008                                 1.03           0.96           1.07           *P* ~A~ = 0.17        *P* ~A~ = 0.19       *P* ~A~ = 0.12
                                                   (28.9)                                               (28.1)                                                      (29.9)                            (0.95--1.11)   (0.89--1.04)   (0.98--1.16)                                             
  AG                                                297                                                  148                                                         168                                  1.22           1.09           1.12           *P* ~D~ = 0.19        *P* ~D~ = 0.15       *P* ~D~ = 0.94
                                                   (29.1)                                               (24.0)                                                      (26.9)                            (1.00--1.49)   (0.90--1.32)   (0.90--1.40)                                             
  GG                                                 21                                                   14                                                          5                                   0.74           2.04           0.37           *P* ~R~ = 0.31        *P* ~R~ = 0.16   ***P*** **~R~ = 0.043**
                                                   (30.8)                                               (42.1)                                                      (15.1)                            (0.37--1.45)   (0.77--5.42)   (0.13--1.04)                                             
  *MMP12* N122S (rs652438)                                                                                                                                                                                                                                                                   
  AA                                                1540                                                 909                                                         926                                  1.01           1.00           1.01       ***P*** **~A~ = 0.021**   *P* ~A~ = 0.23   ***P*** **~A~ = 0.013**
                                                   (28.5)                                               (28.3)                                                      (28.5)                            (0.93--1.10)   (0.93--1.09)   (0.92--1.10)                                             
  AG                                                365                                                  164                                                         220                                  1.32           0.95           1.39       ***P*** **~D~ = 0.022**   *P* ~D~ = 0.33   ***P*** **~D~ = 0.004**
                                                   (30.7)                                               (23.3)                                                      (32.3)                            (1.10--1.59)   (0.80--1.12)   (1.14--1.71)                                             
  GG                                                 29                                                   22                                                          21                                  0.81           0.63           1.28           *P* ~R~ = 0.35        *P* ~R~ = 0.11       *P* ~R~ = 0.52
                                                   (24.8)                                               (30.9)                                                      (39.6)                            (0.47--1.42)   (0.36--1.10)   (0.70--2.32)                                             

*P* ~A~ = *P*-value for additive genetic model, *P* ~D~ = *P*-value for dominant genetic model, *P* ~R~ = *P*-value for recessive genetic model. 

\**P*-value for gene-by-treatment interaction. 

ACM, all-cause mortality; CHL, chlorthalidone treatment group; AML, amlodipine treatment group; LIS, lisinopril treatment group. 

10.1371/journal.pone.0023609.t007

###### Pharmacogenetic effects of MMP genetic variants on stroke. 

![]( pone.0023609.t007){#pone-0023609-t007-7}

                                 Number of events (event rate per 1000 p-y)   Genotype-specific treatment effects: Hazard Ratio (95% CI)   Pharmacogenetic effects[\*](#nt118){ref-type="table-fn"}                                                                                          
  ----------------------------- -------------------------------------------- ------------------------------------------------------------ ---------------------------------------------------------- -------------- -------------- -------------- ------------------------- ---------------- ----------------
  *MMP9* R279Q (rs17576)                                                                                                                                                                                                                                                                     
  AA                                                288                                                  146                                                         180                                  1.15           0.96           1.20           *P* ~A~ = 0.63        *P* ~A~ = 0.29   *P* ~A~ = 0.47
                                                   (10.4)                                               (9.0)                                                       (10.8)                            (0.94--1.40)   (0.80--1.16)   (0.96--1.49)                                             
  AG                                                253                                                  145                                                         185                                  1.05           0.78           1.35           *P* ~D~ = 0.41        *P* ~D~ = 0.18   *P* ~D~ = 0.68
                                                   (8.6)                                                (8.2)                                                       (11.1)                            (0.86--1.29)   (0.65--0.94)   (1.08--1.67)                                             
  GG                                                 70                                                   45                                                          45                                  0.95           0.93           1.02           *P* ~R~ = 0.46        *P* ~R~ = 0.75   *P* ~R~ = 0.34
                                                   (8.9)                                                (9.4)                                                       (9.6)                             (0.65--1.38)   (0.64--1.35)   (0.68--1.55)                                             
  *MMP9* R668Q (rs2274756)                                                                                                                                                                                                                                                                   
  GG                                                456                                                  247                                                         286                                  1.08           0.93           1.16           *P* ~A~ = 0.12        *P* ~A~ = 0.16   *P* ~A~ = 0.15
                                                   (9.8)                                                (9.1)                                                       (10.5)                            (0.93--1.26)   (0.81--1.08)   (0.98--1.37)                                             
  GA                                                139                                                   87                                                         114                                  0.99           0.71           1.39           *P* ~D~ = 0.99        *P* ~D~ = 0.10   *P* ~D~ = 0.15
                                                   (8.3)                                                (8.4)                                                       (11.6)                            (0.76--1.30)   (0.56--0.92)   (1.05--1.84)                                             
  AA                                                 17                                                   3                                                           11                                  3.65           1.04           3.50       ***P*** **~R~ = 0.048**   *P* ~R~ = 0.62   *P* ~R~ = 0.11
                                                   (11.3)                                               (3.1)                                                       (10.7)                            (1.07--12.5)   (0.49--2.23)   (0.98--12.6)                                             
  *MMP12* −82A\>G (rs2276109)                                                                                                                                                                                                                                                                
  AA                                                507                                                  283                                                         356                                  1.06           0.83           1.28           *P* ~A~ = 0.42        *P* ~A~ = 0.18   *P* ~A~ = 0.29
                                                   (9.3)                                                (8.8)                                                       (11.2)                            (0.92--1.23)   (0.73--0.95)   (1.10--1.50)                                             
  AG                                                 98                                                   48                                                          53                                  1.25           1.14           1.09           *P* ~D~ = 0.57        *P* ~D~ = 0.07   *P* ~D~ = 0.29
                                                   (10.1)                                               (8.1)                                                       (8.9)                             (0.88--1.76)   (0.82--1.60)   (0.74--1.62)                                             
  GG                                                 6                                                    5                                                           2                                   0.59           1.58           0.43           *P* ~R~ = 0.31        *P* ~R~ = 0.50   *P* ~R~ = 0.16
                                                   (9.2)                                                (15.8)                                                      (6.1)                             (0.18--1.94)   (0.32--7.81)   (0.08--2.25)                                             
  *MMP12* N122S (rs652438)                                                                                                                                                                                                                                                                   
  AA                                                483                                                  261                                                         315                                  1.10           0.92           1.20           *P* ~A~ = 0.62        *P* ~A~ = 0.40   *P* ~A~ = 0.43
                                                   (9.4)                                                (8.5)                                                       (10.2)                            (0.95--1.28)   (0.80--1.06)   (1.02--1.42)                                             
  AG                                                108                                                   62                                                          83                                  1.04           0.74           1.41           *P* ~D~ = 0.56        *P* ~D~ = 0.22   *P* ~D~ = 0.60
                                                   (9.7)                                                (9.3)                                                       (13.1)                            (0.76--1.42)   (0.56--0.99)   (1.01--1.96)                                             
  GG                                                 11                                                   9                                                           5                                   0.72           1.01           0.71           *P* ~R~ = 0.36        *P* ~R~ = 0.80   *P* ~R~ = 0.32
                                                   (10.2)                                               (14.2)                                                      (10.1)                            (0.30--1.74)   (0.35--2.91)   (0.24--2.11)                                             

*P* ~A~ = *P*-value for additive genetic model, *P* ~D~ = *P*-value for dominant genetic model, *P* ~R~ = *P*-value for recessive genetic model. 

\**P*-value for gene-by-treatment interaction. 

CHL, chlorthalidone treatment group; AML, amlodipine treatment group; LIS, lisinopril treatment group. 

10.1371/journal.pone.0023609.t008

###### Pharmacogenetic effects of MMP genetic variants on CHF. 

![]( pone.0023609.t008){#pone-0023609-t008-8}

                                 Number of events (event rate per 1000 p-y)   Genotype-specific treatment effects: Hazard Ratio (95% CI)   Pharmacogenetic effects[\*](#nt121){ref-type="table-fn"}                                                                                                   
  ----------------------------- -------------------------------------------- ------------------------------------------------------------ ---------------------------------------------------------- -------------- -------------- -------------- ------------------------- ------------------------- ----------------
  *MMP9* R279Q (rs17576)                                                                                                                                                                                                                                                                              
  AA                                                359                                                  283                                                         239                                  0.73           0.90           0.82           *P* ~A~ = 0.53            *P* ~A~ = 0.41        *P* ~A~ = 0.29
                                                   (13.1)                                               (17.8)                                                      (14.5)                            (0.63--0.86)   (0.76--1.06)   (0.69--0.97)                                                      
  AG                                                346                                                  281                                                         255                                  0.73           0.77           0.95           *P* ~D~ = 0.68            *P* ~D~ = 0.20        *P* ~D~ = 0.38
                                                   (11.9)                                               (16.2)                                                      (15.5)                            (0.63--0.86)   (0.65--0.90)   (0.80--1.13)                                                      
  GG                                                 97                                                   95                                                          70                                  0.61           0.82           0.75           *P* ~R~ = 0.26            *P* ~R~ = 0.92        *P* ~R~ = 0.35
                                                   (12.4)                                               (20.2)                                                      (15.2)                            (0.46--0.81)   (0.60--1.11)   (0.55--1.03)                                                      
  *MMP9* R668Q (rs2274756)                                                                                                                                                                                                                                                                            
  GG                                                591                                                  463                                                         403                                  0.74           0.85           0.87           *P* ~A~ = 0.57            *P* ~A~ = 0.59        *P* ~A~ = 0.76
                                                   (12.8)                                               (17.3)                                                      (15.0)                            (0.65--0.83)   (0.75--0.97)   (0.76--0.99)                                                      
  GA                                                192                                                  175                                                         149                                  0.67           0.75           0.90           *P* ~D~ = 0.34            *P* ~D~ = 0.33        *P* ~D~ = 0.93
                                                   (11.5)                                               (17.2)                                                      (15.4)                            (0.55--0.82)   (0.60--0.93)   (0.72--1.12)                                                      
  AA                                                 17                                                   19                                                          14                                  0.57           0.83           0.69           *P* ~R~ = 0.49            *P* ~R~ = 0.97        *P* ~R~ = 0.49
                                                   (11.5)                                               (20.1)                                                      (13.8)                            (0.30--1.10)   (0.41--1.68)   (0.35--1.37)                                                      
  *MMP12* −82A\>G (rs2276109)                                                                                                                                                                                                                                                                         
  AA                                                628                                                  546                                                         467                                  0.67           0.78           0.86           *P* ~A~ = 0.05        ***P*** **~A~ = 0.040**   *P* ~A~ = 0.15
                                                   (11.6)                                               (17.3)                                                      (14.9)                            (0.60--0.76)   (0.69--0.88)   (0.76--0.98)                                                      
  AG                                                154                                                   99                                                          96                                  0.95           0.98           0.97       ***P*** **~D~ = 0.015**   ***P*** **~D~ = 0.044**   *P* ~D~ = 0.77
                                                   (16.0)                                               (17.0)                                                      (16.4)                            (0.74--1.22)   (0.76--1.26)   (0.73--1.28)                                                      
  GG                                                 14                                                   9                                                                                               0.77           3.76           0.21           *P* ~R~ = 0.83        ***P*** **~R~ = 0.048**   *P* ~R~ = 0.07
                                                   (22.4)                                               (28.8)                                                      (6.1)                             (0.33--1.78)   (0.85--16.6)   (0.04--0.95)                                                      
  *MMP12* N122S (rs652438)                                                                                                                                                                                                                                                                            
  AA                                                639                                                  522                                                         450                                  0.72           0.84           0.86           *P* ~A~ = 0.98            *P* ~A~ = 0.41        *P* ~A~ = 0.55
                                                   (12.5)                                               (17.3)                                                      (14.8)                            (0.64--0.81)   (0.75--0.95)   (0.76--0.97)                                                      
  AG                                                131                                                  108                                                          96                                  0.72           0.77           0.93           *P* ~D~ = 0.90            *P* ~D~ = 0.37        *P* ~D~ = 0.46
                                                   (11.7)                                               (16.4)                                                      (15.2)                            (0.55--0.92)   (0.59--1.00)   (0.70--1.22)                                                      
  GG                                                 14                                                   12                                                          12                                  0.68           0.52           1.31           *P* ~R~ = 0.84            *P* ~R~ = 0.24        *P* ~R~ = 0.34
                                                   (12.9)                                               (19.2)                                                      (24.8)                            (0.31--1.46)   (0.24--1.13)   (0.59--2.91)                                                      

*P* ~A~ = *P*-value for additive genetic model, *P* ~D~ = *P*-value for dominant genetic model, *P* ~R~ = *P*-value for recessive genetic model. 

\**P*-value for gene-by-treatment interaction. 

CHF; chronic heart failure; CHL, chlorthalidone treatment group; AML, amlodipine treatment group; LIS, lisinopril treatment group. 

### CHD, combined CHD, and combined CVD {#s3b1}

Participants having the *MMP9* R668Q minor allele homozygote (AA) genotype had lower risk of CCHD when randomized to chlorthalidone versus amlodipine (HR = 0.64).  No significant differences in CHD or CCHD were observed across AG or GG genotype-treatment groups.  The *P*-value for the pharmacogenetic effect for CCHD in the recessive genetic model was 0.038. 

Subjects with the *MMP9* R279Q GG genotype had lower risk of CCVD when randomized to chlorthalidone versus amlodipine or lisinopril (HR = 0.81 and 0.78, respectively) compared with participants with at least one copy of the A allele (pharmacogenetic effect *P* = 0.024 and *P* = 0.026 for chlorthalidone versus amlodipine and chlorthalidone versus lisinopril, respectively in recessive models).  A similar effect was observed for the CCHD outcome, with only the chlorthalidone versus lisinopril comparison reaching a *P*-value\<0.05 (*P* = 0.045 for the recessive model). 

Participants having the *MMP12* N122S variant GG genotype had significantly increased rates (40.9 per 1000 p-y) of CHD if taking lisinopril versus any other genotype-treatment combination (16.3--20.6 per 1000 p-y).  This difference led to a detectable pharmacogenetic effect for both chlorthalidone versus lisinopril and lisinopril versus amlodipine comparisons (*P*-values for pharmacogenetic effect ranged from 0.005 to 0.041 depending on genetic model).  Results for CCHD and CCVD were consistent with this observation. 

For the *MMP12* −82A\>G variant, there was evidence of a pharmacogenetic effect on CCVD for the chlorthalidone versus lisinopril comparison.  Homozygotes for the minor G allele had increased risk of CCVD when randomized to chlorthalidone versus lisinopril (HR = 1.75), while the common A allele carriers had slightly reduced risk (HR = 0.90 for AA group, HR = 0.95 for AG group; pharmacogenetic effect *P* = 0.025). 

### ACM {#s3b2}

Participants with the *MMP12* N122S AG and GG genotypes experienced substantially increased ACM (39% and 28%, respectively) on lisinopril compared to those on amlodipine, while no differences in ACM were found with the common AA genotype-treatment combinations.  The *P*-value for the pharmacogenetic effect was 0.013 when modeled additively and 0.004 when modeled dominantly.  For the chlorthalidone versus amlodipine comparison, GG participants had reduced risk on chlorthalidone (HR = 0.81), while AG heterozygotes had increased risk (HR = 1.32).  There was no treatment effect among the common AA homozygotes (HR = 1.01).  The *P*-value for this pharmacogenetic effect was 0.021 when modeled additively. 

Subjects having the *MMP12* −82A\>G GG genotype had significantly lower ACM on lisinopril versus amlodipine (HR = 0.37), while those with at least one copy of the A allele had somewhat higher risk on lisinopril versus amlodipine (HR = 1.07 and 1.12 for AA and AG groups, respectively; *P* = 0.043 for the pharmacogenetic effect when modeled recessively). 

### Stroke {#s3b3}

Those subjects having *MMP9* R668Q common GG genotype randomized to chlorthalidone had a slightly increased risk of stroke over those with the same genotype randomized to amlodipine (HR = 1.08), whereas participants with the AA genotype had a 3.65-fold increased risk of stroke when randomized to chlorthalidone versus amlodipine.  There was no treatment effect among heterozygotes (HR = 0.99).  The *P*-value for the pharmacogenetic effect was marginally significant (*P* = 0.048). 

### HF {#s3b4}

A pharmacogenetic effect is suggested for the *MMP12* −82A\>G polymorphism with both the chlorthalidone versus amlodipine group and the chlorthalidone versus lisinopril group comparisons.  All subjects with data for the *MMP12* −82A\>G polymorphism, regardless of genotype, showed decreased risk of HF on chlorthalidone versus amlodipine.  However, there was a stronger protective effect of chlorthalidone versus amlodipine among those with the common AA genotype compared to G allele carriers (HR = 0.67 for AA group, HR = 0.95 for AG group, HR = 0.77 for GG group; *P* = 0.015 for pharmacogenetic effect when modeled dominantly).  Among those with the GG genotype, a 3.76-fold increased risk of HF was found for those subjects randomized to chlorthalidone vs. lisinopril, whereas for those with the common AA genotype the risk of HF was reduced in the chlorthalidone group versus the lisinopril group (HR = 0.78).  There was no difference in treatment effect among AG heterozygotes (HR = 0.98).  Although this pharmacogenetic effect is significant (*P* = 0.040 for the additive model, *P* = 0.044 for the dominant model, *P* = 0.048 for the recessive model), it should be noted that there were few events (n = 2) in the GG genotype-lisinopril group. 

Implications of Hardy-Weinberg disequilibrium {#s3c}
---------------------------------------------

Because *MMP12* N122S and *MMP9* R668Q variants were not in HWE for all ethnic subgroups, we tested whether the suggestive pharmacogenetic effects would be similar if we omitted the subgroups in HW disequilibrium.  Therefore, for the *MMP12* N122S analysis we omitted the Black-non-Hispanic participants (31% of the overall population), and for the *MMP9* R668Q analysis we omitted the White-Hispanic participants (13% of the overall population).  The results of these analyses showed that for all the findings presented in [Tables 3](#pone-0023609-t003){ref-type="table"}, [4](#pone-0023609-t004){ref-type="table"}, [5](#pone-0023609-t005){ref-type="table"}, [6](#pone-0023609-t006){ref-type="table"}, [7](#pone-0023609-t007){ref-type="table"}, [8](#pone-0023609-t008){ref-type="table"} for these two variants with a *P*-value less than 0.05, the pharmacogenetic effect (ratio of hazard ratios) was in the same direction and, for the most part, of similar magnitude (data not shown).  Due in part to reduced sample sizes, the *P*-values varied in most cases.  In some cases the association was slightly strengthened.  In cases where association was attenuated, the most disparate finding when omitting the subgroup in HW disequilibrium was found for the pharmacogenetic effect of the *MMP9* R668Q variant on combined CHD when comparing chlorthalidone to amlodipine: for the full group modeled recessively, the ratio of hazard ratios was 0.64 (*P* = 0.04), whereas it was 0.71 (*P* = 0.13) when omitting the White-Hispanic group. 

Discussion {#s4}
==========

In this study we evaluated the pharmacogenetic effects of *MMP9* and *MMP12* variants on CHD, stroke, HF, combined CHD and CVD, and ACM.  Our data provide evidence of pharmacogenetic associations between variants in the *MMP9* and *MMP12* genes and treatment for a variety of these cardiovascular outcomes: *MMP9* R668Q variant for both combined CHD and stroke; *MMP9* R279Q variant for the combined CVD outcome; and *MMP12* N122S variant for CHD, combined CHD, combined CVD, and ACM.  Specifically, we found that patients having the *MMP9* R668Q AA genotype who were treated with chlorthalidone have a decreased risk of combined CHD but an increased risk of stroke compared to patients treated with amlodipine.  Patients with the *MMP9* R279Q GG genotype treated with chlorthalidone had lower risk of combined CVD than patients treated with amlodipine or lisinopril.  For those having either *MMP12* N122S AG or GG genotype, lisinopril treatment increased the risk of CHD, combined CHD, and combined CVD when compared to patients treated with chlorthalidone or a higher risk of CHD and ACM when compared to patients treated with amlodipine.  In aggregate, these results indicate these variants may be useful in selecting appropriate antihypertensive agents to reduce risk of CVD. 

Because some *MMP12* and *MMP9* variants were not in HWE for all ethnic subgroups, we investigated whether the pharmacogenetic effects would be similar if we omitted the subgroups in HW disequilibrium.  These analyses showed that the pharmacogenetic effect was in the same direction and of similar magnitude with the reduced sample.  We note that both the Black and Hispanic groups likely had diverse ancestry given that participants were recruited in the US, Canada, and the Caribbean; therefore, some degree of population substructure is expected.  However, it should be emphasized that even if population substructure in the data leads to HW disequilibrium, which can lead to an increase in false-positive findings for *main effects* of genetic variants on outcomes, this should not explain any *pharmacogenetic findings* because participants were randomized to treatment; therefore, confounding from population substructure should be controlled because randomization in the context of a large sample size results in the same degree of population substructure in *each* treatment group.  We conclude that, although unmeasured population substructure may be present, it is unlikely that the HW disequilibrium in some ethnic subgroups is driving our suggestive results. 

MMP-9 may have a direct effect on plaque destabilization and may also serve as a biomarker predictive of mortality in patients with CVD [@pone.0023609-Blankenberg1].  As previously mentioned, elevated MMP-9 levels have been observed following acute coronary events [@pone.0023609-Kai1]\_ENREF_16.  However, Johnson et al.  suggested MMP-9 is activated during healing processes as opposed to causing rupture of plaques [@pone.0023609-Johnson1].  Based on our findings, chlorthalidone and amlodipine could potentially influence MMP-9 function.  Chlorthalidone, a thiazide-like diuretic, acts on the Na^+^Cl^−^ symporter in the kidney, leading to greatly increased sodium and chloride excretion [@pone.0023609-National1], [@pone.0023609-DrugBank1].  Chlorthalidone\'s antihypertensive mechanism is not fully understood, and we are not aware of studies evaluating independent effects of chlorthalidone on MMP activity.  However, Seeland et al.  examined the effects of diuretic therapy in combination with an ACE inhibitor and found supplemental treatment with the diuretic furosemide did not provide additional benefit on extracellular matrix remodeling compared to ACE inhibitor alone [@pone.0023609-Seeland1].  Amlodipine is a CCB of the dihydropyridine class.  It inhibits vascular smooth muscle contractions, causing increased blood flow to the heart and decreased peripheral arterial resistance and blood pressure [@pone.0023609-DrugBank2].  Zervoudaki et al.  reported that amlodipine increases plasma MMP-9 levels in hypertensive patients [@pone.0023609-Zervoudaki1], [@pone.0023609-Zervoudaki2].  However, a study involving lercanidipine (another dihydropyridine CCB) found decreased MMP-9 activity in hypertensives and suggested a mechanism involving the antioxidant effects of lercanidipine, which are shared by amlodipine [@pone.0023609-Martinez1].  A possible explanation for this discrepancy is that the amlodipine study used ELISA to measure MMP-9 level, whereas the more recent lercanidipine study used gel zymography [@pone.0023609-Martinez1].  Like other ACE inhibitors, lisinopril blocks the production of angiotensin II (a vasoconstrictor), thereby decreasing blood pressure.  Like the MMPs, ACE is a zinc-dependent endopeptidase [@pone.0023609-Jin1].  Studies have shown that ACE inhibition may also inhibit MMP levels [@pone.0023609-Jin1], [@pone.0023609-Reinhardt1], [@pone.0023609-Yamamoto1], [@pone.0023609-Liebetrau1], [@pone.0023609-Takai1], [@pone.0023609-Yamamoto2], [@pone.0023609-Yamamoto3].  Yamamoto et al.  identified two potential interaction mechanisms between lisinopril and the active site of MMP-9 [@pone.0023609-Yamamoto3].  Lisinopril was shown to be stabilized in the active site by specific hydrogen bonds, and its hydrophobic group interacted preferentially with the S1 site compared to the S1′ site [@pone.0023609-Yamamoto3].  In their subsequent work, Yamamoto et al.  and Takai et al.  also found that different ACE inhibitors show differential binding affinities for MMP-9 [@pone.0023609-Yamamoto1], [@pone.0023609-Takai1], [@pone.0023609-Yamamoto2], [@pone.0023609-Yamamoto3].  Sakata et al.  observed that ACE inhibition directly lowers MMP activity in rats, thus preventing left ventricular remodeling [@pone.0023609-Sakata1].  The *MMP9* R279Q polymorphism is a glutamine to arginine substitution located in the catalytic domain of MMP-9 [@pone.0023609-Blankenberg1], [@pone.0023609-Hirose1] and may affect substrate binding [@pone.0023609-Blankenberg1].  The *MMP9* R668Q polymorphism is in the hemopexin-like domain and probably also functions in substrate binding, since removal of this domain disables the cleavage of triple-helix collagen [@pone.0023609-Han1]. 

MMP-12 is expressed in macrophages and epithelial cells and has been implicated in the progression of atherosclerosis, wound repair, and certain cancers [@pone.0023609-Kerkela1], [@pone.0023609-Lavigne1].  Like MMP-9, MMP-12 activity is increased following vascular injury [@pone.0023609-Matsuno1].  Plasma MMP-12 levels are increased in patients with coronary artery disease and may, therefore, be an independent risk factor for coronary artery disease [@pone.0023609-JguirimSouissi1].  Morgan et al.  observed higher MMP-12 levels in thin-cap atherosclerotic plaques (those considered more prone to rupture) versus thick-cap plaques.  MMP-12 levels were also elevated in already ruptured plaques, suggesting that MMP-12 has a role in plaque stability [@pone.0023609-Morgan1].  Based on our findings, chlorthalidone, lisinopril, and amlodipine could potentially affect MMP-12 activity.  To our knowledge, studies have not evaluated the effects of diuretics, ACE inhibitors, or CCBs on MMP-12.  However, based on the finding that lercanidipine may inhibit macrophage function and the fact that macrophages secrete MMP-12, we hypothesize that CCBs may decrease MMP-12 levels [@pone.0023609-Kerkela1], [@pone.0023609-Canavesi1].  The MMP12 −82A\>G polymorphism lies in the *MMP12* gene promoter [@pone.0023609-Jormsjo1].  The *MMP12* −82A\>G polymorphism affects AP-1 binding affinity, with the A allele showing a higher affinity for the protein and, therefore, higher *MMP12* promoter activity [@pone.0023609-Jormsjo1], [@pone.0023609-Joos1]. * MMP12* N122S is an asparagine to serine substitution located in the coding region of the *MMP12* hemopexin domain, which is responsible for gene activity [@pone.0023609-Su1], [@pone.0023609-Joos1].  To our knowledge, no pharmacogenetic studies have been done on this combination of polymorphisms, drugs, and outcomes. 

Our data suggest associations between the *MMP9* R279Q polymorphism and stroke and between the *MMP12* N122S polymorphism and combined CHD.  The *MMP9* R279Q polymorphism has been associated with increased intima-media thickness [@pone.0023609-Armstrong1], an independent predictor of MI and stroke risk [@pone.0023609-Bots1].  Blankenberg et al.  found that *MMP9* R279Q had no effect on plasma MMP-9 concentration but was associated with future cardiovascular events in patients with stable angina [@pone.0023609-Blankenberg1].  This polymorphism also influences aortic stiffness, which is a determinant of cardiovascular risk [@pone.0023609-Yasmin1].  We also found a pharmacogenetic effect of the *MMP9* R668Q polymorphism to increased stroke risk, although this association may be due to the small number of events (17 in the chlorthalidone group, 3 in the amlodipine group).  The *MMP12* −82A\>G polymorphism has been associated with coronary atherosclerosis [@pone.0023609-Jormsjo1], [@pone.0023609-Ye1], [@pone.0023609-Ye2].  A study by Jormsjo et al.  suggests this polymorphism is associated with narrowing of coronary arteries in diabetic patients with CHD [@pone.0023609-Jormsjo1], [@pone.0023609-Ye2]. 

GenHAT is an ancillary study of ALLHAT.  The ALLHAT study population included only those over age 55 with hypertension.  As a result, our findings may not be generalizable to younger age groups.  An additional limitation is that we looked at only four MMP polymorphisms.  There may be unknown polymorphisms in these genes or combinations of polymorphisms affecting the findings.  Since we performed multiple tests of pharmacogenetic effects, these findings would not meet the threshold of statistical significance if corrected for multiple testing (Bonferroni correction: 0.05/72 tests would equate to a *P*-value of 0.0007).  However, given the possibility of linkage disequilibrium between variants and non-mutually exclusive outcomes, this threshold is overly conservative.  Nevertheless, the likelihood that these findings are false positives is not negligible, and independent replication is necessary.  At this time, there are no other large clinical trials that are outcome-based like ALLHAT in which results can be replicated.  Our findings and the findings of other studies suggest that future studies might fruitfully investigate the functional interactions of MMP-9 and MMP-12 with diuretics, ACE inhibitors, and CCBs.  This study has several strengths.  The ALLHAT trial was a large, double-blind randomized trial.  Additionally, the study population showed exceptional ethnic and gender diversity (about 50% non-White, about 50% female) [@pone.0023609-Arnett1].  Our results were not sensitive to departures from HWE in various ethnic groups. 

**Competing Interests:**BD has twice served as a consultant on the Takeda Pharmaceuticals Data Safety Monitoring Board and has received one honoraria for work done for Forest Laboratories.  VB is employed by Roche Molecular Systems, Inc., a company with no current business interests in cardiovascular disease.  These potential competing interests in no way compromise the authors\' adherence to PLoS ONE data and material sharing policies.  All other authors disclosed no potential conflicts of interest. 

**Funding:**This study was supported by grants HL63082 (Genetics of Hypertension Associated Treatments, (GenHAT)) from the National Heart, Lung, and Blood Institute (<http://www.nhlbi.nih.gov>) and N01-HC-35130 (Antihypertensive and Lipid-Lowering Treatment to Prevent Heart Attack Trial, (ALLHAT)) from the National Institutes of Health (<http://www.nih.gov>).  The National Heart, Lung, and Blood Institute had a contributing role in the analysis and interpretation of the ALLHAT data and in the review and approval of this GenHAT article. 

[^1]: Conceived and designed the experiments: JE BD CF EB DA.  Performed the experiments: EB.  Analyzed the data: RT AL.  Contributed reagents/materials/analysis tools: VB.  Wrote the paper: RT DA AL.