Studies with MMP9 gene promoter polymorphism and nonsyndromic cleft lip and palate^†

To the Editor:

Nonsyndromic cleft lip with or without palate (CL/P) is one of the most common congenital defects in humans. Birth prevalence varies based on ethnicity and geographic location [Vanderas, 1987]. Etiology still remains unclear whilst the molecular mechanisms that control palate development are complex and not thoroughly understood.

Palatogenesis requires remodeling of the extracellular matrix (ECM) and subsequent fusion of the palatal shelves. In normal physiologic conditions, ECM remodeling is a strictly controlled process necessary for embryonic development and organogenesis [Woessner, 1994]. Disruption of the coordinated migration and fusion of facial processes by genetic, environmental, or combined factors at any time point could lead to CL/P [Wong and Hägg, 2004].

Matrix metalloproteinases (MMPs) comprise a group of likely candidate proteins involved in the etiology of CL/P because of their role in modeling craniofacial tissues [Iamaroon et al., 1996]. Temporospatial expression of MMPs 2, 3, 7, 9, and 13 was observed during murine palatal fusion [Morris-Wiman et al., 1999, 2000]. Moreover, previous studies have demonstrated an involvement of MMPs in the developing palate implying that such process requires proteolytic degradation of ECM, and MMPs as a necessary step for lip and palatal fusion [Morris-Wiman et al., 2000; Blavier et al., 2001; Brown et al., 2002]. MMP9 gene maps to chromosome 20q12.2 and is known for its ability in degrading type IV collagen, a main component of the ECM, and facilitating cell migration [Zhang et al., 1999]. Blavier et al. 2001 detected a selective expression of MMP-9 in the ossification centers in the midline of the developing maxilla; however, nothing has ever been mentioned known about a role of MMP9 in the occurrence of CL/P. Nevertheless, the finding that degradation of the basal membrane adjacent to the medial epithelial edge (MEE) occurs simultaneously to epithelial-mesenchymal transformation (EMT), and the overexpression of certain MMPs in the MEE between the palatal shelves during fusion suggest that they might somehow be involved [Kaartinen et al., 1997; Blavier et al., 2001; Brown et al., 2002; Kang and Svoboda, 2005].

Polymorphisms in genes including TGFα [Ardinger et al., 1989; Machida et al., 1999; Jugessur et al., 2003], MSX1 [Jezewski et al., 2003; Vieira et al., 2003], and TGF-β3 [Jugessur et al., 2003; Vieira et al., 2003] have been associated to CL/P in nonsyndromic patients. A polymorphism on chromosome 16p13, comprising MMP25 gene, has also yielded evidence for linkage and association with CL/P [Blanton et al., 2004].

A polymorphism on the MMP9 gene promoter (−1562 C/T) causes a functional effect on transcription, in which a C to T substitution results in the loss of binding of a nuclear protein to this region of the MMP9 gene and an increase in transcriptional activity [Zhang et al., 1999]. Palate EMT requires several steps for the two epithelial sheets to fuse at the apical membranes and some cells may move to the oral or nasal epithelium. The cells increase, among other signaling molecules, in MMPs, for matrix degradation [Kang and Svoboda, 2005]. A decrease in MMP levels could hypothetically lead to failure of fusion and a cleft of the lip and/or palate.

To address this hypothesis, genomic DNA samples from 125 nonsyndromic cleft lip/palate individuals ascertained through the Hospital of Rehabilitation and Craniofacial Anomalies of the University of São Paulo, Bauru, SP, Brazil and 173 healthy control individuals were analyzed by PCR-RFLP and gel electrophoresis. The study was approved by the IRB of the University of São Paulo and by the Brazilian Council for Ethics and Research on Human Subjects. All patients signed an informed consent sheet in agreement to the terms of the study.

Basic parameters of the population studied are shown on Table I. Genotypes were detected by polymerase chain reaction-restriction fragment length polymorphism. (PCR-RFLP). PCR was carried out in a total volume of 25 µl containing 50 ng genomic DNA, 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl₂, 1 µM of each primer, 200 mM dNTPs, and 2.5 units Taq DNA polymerase (Invitrogen, Carlsbad, CA). Primer sequences were 5′-GCCTGGCACATAGTAGGCCC-3′ (forward) and 5′-CTTCCTAGCCAGCCGGCATC-3′ (reverse). Reaction cycles were 95°C for 3 min, 35 cycles at 95°C for 1 min, 65°C for 45 sec, 72°C for 45 sec, and final extension at 72°C for 7 min. Enzyme digestion was realized adding 2 µl of PCR products to a 8 µl solution containing 1 µl 10X NE buffer, 0.3 µl Sph I (20 units/ml) (New England Biolabs, Ipswich, MA), and 6.7 µl sterile deionized water at 37°C overnight. The total amount of the digest was electrophoresed on a 10% vertical nondenaturing polyacrylamide gel at 20 mA. Staining procedures followed previously published protocols [Sanguinetti et al., 1994].

We tested the association between the MMP9 −1562 C/T polymorphism and CL/P using a case-control design. Differences in frequencies of the polymorphism in both groups were assessed by Chi-square test (χ²) and using the odds ratio and 95% confidence intervals. Statistically significant differences were set at P < 0.05. The distribution of the MMP9 genotypes in cases and controls was consistent with Hardy–Weinberg equilibrium (P > 0.05; Table II). We found no evidence for genotypic association in any of the groups (OR = 1.307, 95% CI: 0.74–2.30; Table II). The TT genotype which is believed to increase MMP-9 transcription and reported to be very low was not observed in any of the groups. No significant differences were observed regarding allele frequencies in any of the groups (P > 0.05).

Logistic regression showed no differences in genotype distribution between cases and controls adjusted by race and gender (OR = 1.3821, 95% CI: 0.73–2.60, and OR = 1.6193, 95% CI: 0.93–2.82, respectively). The Brazilian population consists of a large ethnic mixture of Latin-European whites, Afro-Brazilian blacks, and Amerindians, which makes it very difficult to match perfectly the ethnicity of cases and controls and may have accounted for some confounding effects of undetectable population stratification. However, the discrete difference in the frequency of the T allele between cases and controls seems not to be enough to account for such effect. In an attempt to overcome the problem of population stratification, since most individuals in this study were of Caucasian ethnicity, we compared genotype and allele frequencies in Caucasians only, and observed no significant differences (OR = 1.11, 95% CI: 0.57–2.13 and OR = 1.09, 95% CI: 0.59–2.03, for genotype and allele frequencies, respectively).

Somewhat interesting results were found when cleft lip only, cleft lip with cleft palate and cleft palate only were evaluated separately (Table III). The CL group did not present significant differences regarding genotype distribution, but CLP and CP groups presented significantly more CC genotypes than CT (P < 0.05) (Table III). In addition, the CLP group showed differences in genotype distribution and gender. Male cases frequently presented more CC genotypes than females; the opposite was true for the CT genotype (OR = 2.57, 95% CI: 1.00–6.61).

Our hypothesis that CL/P individuals would tend to present significantly more CC genotypes than controls, indicating a possible downregulation of the MMP9 gene promoter thus decreasing tissue remodeling and further leading to a possible oral cleft was not confirmed. Perhaps this lack of association could be a result of sample size. A convenient sample of 500 cases and 500 controls should achieve 80% statistical power [Purcell et al., 2003]. In addition, other factors such as occupational exposure and certain dietary components might interact with the MMP-9 genotype or act as potential confounders in the analysis. Unfortunately, information on these factors in our case-control study was not available. It would be interesting to investigate the interaction between MMP-9 genotypes and these risk factors in future studies.

Although the results for the MMP9 −1562 C/T polymorphism in this particular study do not provide evidence of a major role for this gene in NSCLP, other MMPs might present positive levels of involvement and their association with CL/P is yet to be elucidated.