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13. Kru¨ ger A, Pu¨ schel K. Haptoglobin subtypes in Northern Germany (Hamburg). Int J Leg Med 1993;106:53– 4. 14. Mastana SS, Fisher P. Haptoglobin subtypes in the East Midlands (United Kingdom). Int J Leg Med 1994;107:52– 4. 15. Loftus BJ, Kim U-J, Sneddon VP, Kalush F, Brandon R, Fuhrmann J, et al. Genome duplications and other features in 12 Mb of DNA sequence from human chromosome 16p and 16q. Genomics 1999;60:295–308. 16. Erickson LM, Kim HS, Maeda N. Junctions between genes in the haptoglobin gene cluster of primates. Genomics 1992;14:948 –58. 17. Chapelle JP, Albert A, Smeets JP, Heusghem C, Kulbertus HE. Effect of the haptoglobin phenotype on the size of a myocardial infarct. N Engl J Med 1982;307:457– 63. 18. Levy AP, Roguin A, Hochberg I, Herer P, Marsh S, Nakhoul FM, et al. Haptoglobin phenotype and vascular complications in patients with diabetes [Letter]. N Engl J Med 2000;343:969 –70. 19. Nakhoul FM, Marsh S, Hochberg I, Leibu R, Miller BP, Levy AP. Haptoglobin genotype as a risk factor for diabetic retinopathy [Letter]. JAMA 2000;284: 1244 –5. 20. Roguin A, Hochberg I, Nikolsky E, Markiewicz W, Meisel SR, Hir J, et al. Haptoglobin phenotype as a predictor of restenosis after percutaneous transluminal coronary angioplasty. Am J Cardiol 2001;87:330 –2. 21. Roguin A, Ribichini F, Ferrero V, Matullo G, Herer P, Wijns W, et al. Haptoglobin phenotype and the risk of restenosis after coronary artery stent implantation. Am J Cardiol 2002;89:806 –10. DOI: 10.1373/clinchem.2003.022442
Colorectal Carcinoma Susceptibility and Metastases Are Associated with Matrix Metalloproteinase-7 Promoter Polymorphisms, Giorgio Ghilardi,1* Maria Luisa Biondi,2 Maddalena Erario,2 Emma Guagnellini,2 and Roberto Scorza1 (1 Dipartimento MCO, Clinica Chirurgica Generale, Universita` degli Studi di Milano, Polo S. Paolo, Via A. Di Rudinı` 8, I-20142 Milan, Italy; 2 Laboratorio di Chimica Clinica e Microbiologia, Ospedale S. Paolo, Polo Universitario, Via A. Di Rudinı` 8, I-20142 Milan, Italy; * author for correspondence: fax 39-02-8137613, e-mail giorgio.
[email protected]) Tumor invasion and metastasis are important aspects of tumor progression, and the formation of tumor metastasis is a principal contributing factor to cancer morbidity and mortality (1 ). Basal membrane and extracellular matrix represent two physical barriers to malignant invasion: their degradation by matrix metalloproteinases (MMPs) plays a key role in tumor progression and metastatic spread (2, 3 ). MMP expression in tumors is regulated in a paracrine manner by growth factors and cytokines secreted by tumor-infiltrating inflammatory cells as well as by tumor or stromal cells. Recent studies have suggested continuous cross-talk between tumor cells, stromal cells, and inflammatory cells during the invasion process (1, 2, 4, 5 ). Expression of most MMPs is usually low in tissues and is induced when remodeling of extracellular matrix is required. MMP gene expression is primarily regulated at the transcriptional level, but there is also evidence of modulation of mRNA stability in response to growth factor and cytokines (2, 6 ). Several lines of evidence indicate a significant association between variations in MMP genes and susceptibility to cancer. The promoter region of inducible MMP genes (i.e., MMP1 and MMP3) shows remarkable conservation
of regulatory elements, and their expression is induced by growth factors, cytokines, and other environmental factors, such as contact with extracellular matrix (7–9 ). Recently, a naturally occurring sequence variation in the human MMP1 gene promoter was reported (10 ). Many studies have demonstrated the correlation between the 2G allele and several malignant tumors with different histogenetic origins (11–15 ). Recently, our group demonstrated a correlation between the MMP3 polymorphism and breast cancer (16 ). Matrilysin (MMP7) is a protease with broad substrate specificity, being able to degrade elastin, proteoglycans, fibronectin, and type IV collagen. MMP7 is among the smallest members of the MMP family (17 ). MMP7 was first characterized from a human rectal carcinoma cell line, and overexpression of MMP7 has been shown to correlate with Duke’s stage and increased metastatic potential in colorectal carcinoma (CRC) (18, 19 ). Jormsjo¨ et al. (20 ) recently described two common polymorphisms in the promoter region of the MMP7 gene that are functional in vitro and seem to influence coronary artery dimensions. Both polymorphisms influenced the binding of nuclear proteins. Furthermore, in transient transfection studies, the combination of the two rare alleles conferred an increased promoter activity. The aim of this prospective study was to investigate a possible correlation between MMP7 promoter polymorphisms and CRC clinical phenotypes, specifically the ability of genetic analysis to identify a subgroup of CRC patients with a disease that appears more aggressive or prone to metastasis. The study started during June 2000, and recruitment was stopped during June 2002. Our Institutional Ethical Committee approved this study, and informed consent was obtained from patients and controls. MMP7 gene promoter sequences were obtained from peripheral blood samples from 58 consecutive patients with CRC of different stages who underwent surgery and from 111 sex- and age-matched healthy individuals (control group). CRC patients were 38 males and 20 females (median age, 68 years; range, 32– 88 years). Patients and controls were all Italian. CRC patients were grouped according to Duke’s classification, as modified by Astler and Coller (21 ), on the basis of the postoperative histopathologic evaluation. The group assignment was then reevaluated at the end of the follow-up period, which ranged from 6 to 30 months (median, 21 months). The 58 patients with CRC were assigned to two subgroups according to the presence (M⫹) or absence (M⫺) of detectable metastasis at the time of diagnosis and at the end of the follow-up. Genetic polymorphisms were detected with PCR followed by direct sequencing. The Insta Gene (Bio-Rad) commercial reagent set was used for DNA extraction from whole blood. The PCR reaction for MMP7 was carried out in a total volume of 25 L with 5 L of extracted genomic DNA; 100 M dATP, dGTP, dTTP and dCTP; 1.5 mM MgCl2; 1 U of Taq Gold polymerase; and the two primers,
Clinical Chemistry 49, No. 11, 2003
forward and reverse, each at a concentration of 80 nM. The primers were designed with the Primer Express software. The MMP7 primer sequences were as follows: forward primer, 5⬘-CCTGAATGATACCTATGAGAGCAGTC-3⬘; reverse primer, 5⬘-AGAGTCTACAGAACTTTGAAAGTATGTGTTATT-3⬘. The PCR began with a 5-min incubation at 94 °C to activate the enzyme, followed by 35 cycles of 20 s at 94 °C, 20 s at 55 °C, and 30 s at 72 °C. The amplification was verified on an agarose gel (2%) followed directly by sequencing with an automatic sequencer using fluorescent DNA capillary electrophoresis (ABI Prism 310; Applied Biosystems). The forward primer was used for the sequencing primer. Differences between groups were examined by the 2 test or Fisher exact test when appropriate. Odds ratios (ORs; approximate relative risk) were calculated as an index of the association of the MMP genotypes with each phenotype. For each OR, two-tailed probability values and 95% confidence intervals (CIs) were calculated. P ⬍0.05 was assumed as the cutoff for statistical significance. All statistical analyses were two-sided and were performed with Stata Statistical Software (Stata Corporation). The polymorphism distributions in patients and controls were, as expected, according to Hardy–Weinberg equilibrium. In CRC patients, the number of ⫺181G homozygotes [patients vs controls, 15 (26%) vs 14 (13%); OR, 2.41; 95% CI, 0.98 –5.89; P ⫽ 0.03] and the T allele frequency in position ⫺153 (0.11 vs 0.05; OR, 2.20; 95% CI, 0.89 –5.48; P ⫽ 0.05) were significantly higher than in controls. The ⫺181G/⫺153T haplotype was more represented, although not significantly, in CRC patients than in controls (P ⫽ 0.08; Table 1). Subgroups were compared with controls and among allelic variants. The ⫺153C/T promoter allelic variant distribution was not statistically significantly different among the subgroups. For the ⫺181A/G promoter polymorphism, however, we found a strong correlation between ⫺181G homozygosity and the M⫹ subgroup at the time of diagnosis [M⫹ vs M⫺, 9 (56%) vs 6 (14%); OR, 7.5; 95% CI, 2.07–27.19; P ⫽ 0.0013]. The frequency of the ⫺181G allele was not statistically different between controls and metastasis-free (M⫺) patients (P ⫽ 0.95). Moreover, the ⫺181A/G polymorphism was correlated with lymph node metastasis at the beginTable 1. Distribution of the ⴚ181G/ⴚ153T haplotype in patients and controls. Subgroups
1 2 3 4 5a 6a 7a
ⴚ181A/G
ⴚ153 C/T
CRC patients, n (%)
Controls, n (%)
AA AG GG AA AG GG GG
CC CC CC CT CT CT TT
14 (24) 21 (36) 11 (19) 1 (2) 7 (12) 3 (5) 1 (2)
35 (31) 54 (49) 10 (9) 1 (1) 7 (6) 4 (4) 0
a P ⫽ 0.08 for subgroups 1– 4 vs 5–7 (presence of both the ⫺181G allele and the ⫺153T mutation).
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ning of follow-up. In the N1⫹N2 patients, the number of ⫺181G homozygotes and G allele frequencies were greater than in the N0 patients [number of homozygotes, 13 (35%) vs 2 (10%; P ⫽ 0.03); G allele frequency, 0.53 vs 0.36 (P ⫽ 0.02), respectively]. Our data demonstrate, for the first time, that the two common polymorphisms on MMP7 promoter region are correlated to CRC susceptibility. In addition, our results may suggest that ⫺181G homozygosity is associated with the presence of distant metastasis as well as lymph node involvement. From this point of view, G homozygosity might be considered as a factor of worse prognosis. MMP7 has unique characteristics, such as a minimum MMP structure, wide spectrum of substrate specificity, and potency to start an activation cascade of MMPs (17 ). In addition to its overexpression in a variety of cancer tissues, in vitro and animal data suggest that MMP7 may play a key role in tumor invasion and metastasis as well as tumor initiation and growth. Many studies have reported that MMP7 mRNA is overexpressed in human CRC and is correlated with increasing Duke’s stage (22–24 ). Recently Jormsjo¨ et al. (20 ) identified two novel polymorphisms in the MMP7 promoter region. Electrophoretic mobility shift assay results demonstrated that the ⫺181A/G and the ⫺153C/T polymorphisms influence the binding of nuclear protein(s). Furthermore, basal promoter activity was higher in promoter constructs harboring the combination of the two rare alleles in transient transfection studies. In our study the ⫺181G/ ⫺153T haplotype was more represented in CRC patients than in controls, although statistical significance was not reached (19% vs 10%; P ⫽ 0.08). We did observe a statistically significant correlation between the ⫺181A/G substitution and susceptibility, invasiveness, and prognosis in CRC. In CRC patients, the frequency of ⫺181G homozygotes was 2-fold higher than in controls, and 4.2-fold higher in patients who developed metastasis at the end of the follow-up (OR, 4.2; 95% CI, 1.19 –14.59; P ⫽ 0.024). Lymph node metastasis is the most important prognostic factor in colon cancer, and the ⫺181A/G substitution correlated well with lymph node involvement at the time of diagnosis. The G allele frequency was 2.5-fold higher in lymph node-positive patients than in lymph node-negative ones (OR, 2.49; 95% CI, 1.14 –5.42; P ⫽ 0.02). The ⫺153C/T polymorphism seems in our series less involved in CRC susceptibility, although the mutant T allele was more represented in CRC patients (P ⫽ 0.05). In conclusion, our study suggests that the presence of G allele for the MMP7 gene promoter sequence may be a facilitating factor for cancer growth, lymph node invasion, and metastasis in CRC patients. The good correlation of lymph node involvement and distant metastasis with ⫺181G homozygosity might suggest the use of this common polymorphism as a prognostic factor at the beginning of follow-up for influencing the decision for adjuvant therapy in N0M0 patients. Our data suggest a role of MMP7 in the matrix remodeling associated with CRC. However, no firm conclusions regarding the clinical sig-
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nificance of MMP7 promoter polymorphisms can be drawn until much larger cohorts have been analyzed. References 1. Basset P, Okada A, Chenard MP, Kannan R, Stoll I, Anglard P, et al. Matrix metalloproteinases as stromal effectors of human carcinoma progression therapeutical implications. Matrix Biol 1997;15:535– 41. 2. Johnsen M, Lund LR, Romer J, Almholt K, Danø K. Cancer invasion and tissue remodeling common themes in proteolytic matrix degradation. Curr Opin Cell Biol 1998;10:667–71. 3. Nelson AR, Fingleton B, Rothenberg ML, Matrisian LM. Matrix metalloproteinases: biologic activity and clinical implications. J Clin Oncol 2000;18: 1135– 49. 4. Urı´a JA, Ståhle-Ba¨ckdahl M, Seiki M, Fueyo A, Lo´pez-Otı´n C. Regulation of collagenase-3 expression in human breast carcinomas is mediated by stromal-epithelial cell interaction. Cancer Res 1997;57:4882– 8. 5. Johansson N, Vaalamo M, Gre´nman S, Hietanen S, Klemi P, Saarialho-Kere U, et al. Collagenase-3 (MMP13) is expressed by tumor cells in invasive vulvar squamous cell carcinomas. Am J Pathol 1999;154:469 – 80. 6. Skobe M, Fusening NE. Tumorigenic conversion of immortal human keratinocytes through small cell activation. Proc Natl Acad Sci U S A 1998;95:1050 –5. 7. Shapiro SD. Matrix metalloproteinase degradation of extracellular matrix: biological consequences. Curr Opin Cell Biol 1998;10:602– 8. 8. Vincenti MP, White LA, Schroen DJ, Benbow U, Brinckerhoff CE. Regulating expression of the gene for matrix metalloproteinase-1 (collagenase): mechanisms that control enzyme activity, transcription, and mRNA stability. Crit Rev Eukaryot Gene Exp 1996;6:391– 411. 9. Wernert N, Gilles F, Faufer V, Bouali F, Raes MB, Pyke C, et al. Stromal expression of c-Ets1 transcription factor correlates with tumor invasion. Cancer Res 1994;54:5683– 8. 10. Rutter JL, Mitchell TI, Buttice´ G, Meyers J, Gusella JF, Ozelius LJ, et al. A single nucleotide polymorphism in the matrix metalloproteinase-1 promoter creates an Ets binding site and augments transcription. Cancer Res 1998;58:5321–5. 11. Ghilardi G, Biondi ML, Mangoni J, Leviti S, DeMonti M, Guagnellini E, et al. Matrix metalloproteinase-1 promoter polymorphism 1G/2G is correlated with colorectal cancer invasiveness. Clin Cancer Res 2001;7:2344 – 6. 12. Kanamori Y, Matsushima M, Minaguchi T, Kobayashi K, Sagae S, Kudo R, et al. Correlation between expression of the matrix metalloproteinase-1 gene in ovarian cancers and an insertion/deletion polymorphism in its promoter region. Cancer Res 1999;59:4225–7. 13. Nishioka Y, Kobayashi K, Sagae S, Ishioka SI, Nishikawa A, Matsushima M, et al. A single nucleotide polymorphism in the matrix metalloproteinase-1 promoter in endometrial carcinomas. Jpn J Cancer Res 2000;91:612–5. 14. Ye S, Dhillon S, Turner SJ, Bateman AC, Theaker JM, Pickering RM, et al. Invasiveness of cutaneous malignant melanoma is influenced by Matrix Metalloproteinase 1 gene polymorphism. Cancer Res 2001;61:1296 – 8. 15. Zhu Y, Spitz MR, Lei L, Mills GB, Wu X. A single nucleotide polymorphism in the matrix metalloproteinase-1 promoter enhances lung cancer susceptibility. Cancer Res 2001;61:7825–9. 16. Ghilardi G, Biondi ML, Caputo M, Leviti S, De Monti M, Guagnellini E, et al. A single nucleotide polymorphism in the matrix metalloproteinase-3 promoter enhances breast cancer susceptibility. Clin Cancer Res 2002;8:3820 –3. 17. Wilson CL, Matrisian LM. Matrilysin: an epithelial matrix metalloproteinase with potentially novel functions. Int J Biochem Cell Biol 1996;28:123–36. 18. Mori M, Barnard GF, Mimori K, Ueo H, Akiyoshi T, Sugimachi K. Overexpression of matrix metalloproteinase-7 mRNA in human colon carcinomas. Cancer 1995;75:1516 –9. 19. Yamamoto H, Itoh F, Hinoda Y, Imai K. Suppression of matrilysin inhibits colon cancer invasion in vitro. Int J Cancer 1995;61:218 –22. 20. Jormsjo¨ S, Whatling C, Walter DH, Zeiher AM, Hamsten A, Eriksson P. Allele-specific regulation of matrix metalloproteinase-7 promoter activity is associated with coronary artery luminal dimensions among hypercholesterolemic patients. Arterioscler Thromb Vasc Biol 2001;21:1834 –9. 21. Chang AE. Colorectal cancer. In: Greenfield LI, ed. Surgery: scientific principles and practice. Philadelphia: JB Lippincott Co., 1993:1014 –31. 22. Ichikawa Y, Ishikawa T, Momiyama N, Yamaguchi S, Masui H, Hasegawa S, et al. Detection of regional lymph node metastases in colon cancer by using RT-PCR for matrix metalloproteinase 7, matrilysin. Clin Exp Metastasis 1998;16:3– 8. 23. Masaki T, Matsuoka H, Sugiyama M, Abe N, Goto A, Sakamoto A, et al. Matrilysin (MMP-7) as a significant determinant of malignant potential of early invasive colorectal carcinomas. Br J Cancer 2001;84:1317–21. 24. NewellKJ, Matrisian LM, Driman DK. Matrilysin (matrix metalloproteinase-7) expression in ulcerative colitis-related tumorigenesis dagger. Mol Carcinog 2002;34:59 – 63. DOI: 10.1373/clinchem.2003.018911
Clinical Evaluation of a Reverse Hybridization Assay for the Molecular Detection of Twelve MEFV Gene Mutations, Dimitri Tchernitchko,1* Marie Legendre,1* Andre´e Delahaye,1 Ce´cile Cazeneuve,2 Florence Niel,1 Michel Goossens,1,2 Serge Amselem,1,2 and Emmanuelle Girodon1† (1 Service de Biochimie et de Ge´ ne´ tique Mole´ culaire, Hoˆ pital Henri Mondor, AP-HP, 94010 Cre´ teil, France; 2 INSERM U468 Ge´ ne´ tique Mole´ culaire et Physiopathologie, 94010 Cre´ teil, France; * the two first authors contributed equally to this work; † address correspondence to this author at: Service de Biochimie et de Ge´ ne´ tique Mole´ culaire– INSERM U468, Hoˆ pital Henri Mondor, 51, Avenue du Mare´ chal de Lattre de Tassigny, 94010 Cre´ teil, France; fax 33-1-4981-2842, e-mail
[email protected]) Familial Mediterranean fever (FMF) is an autosomalrecessive disorder (MIM 249100) characterized by recurrent attacks of fever and serositis, affecting principally Sephardic Jewish, Armenian, Arab, and Turkish populations. Early diagnosis is important to initiate colchicine therapy, which prevents the occurrence of attacks and of renal amyloidosis, the major complication of the disease. The identification of MEFV (1, 2 ), the gene responsible for the disease, allowed the use of an early molecular test of diagnostic value for FMF patients (3 ), which negates the needs for unnecessary invasive investigations. The MEFV gene, located on chromosome 16p13.3, contains 10 exons and encodes the marenostrin/pyrin protein, a molecule acting as a regulator of the proinflammatory interleukin1-dependent pathway and of the apoptosis mediated by the apoptosis-associated Speck-like protein containing a CARD (ASC) (4, 5 ), and which belongs to the death domain-fold family (6 ). More than 40 different FMF-associated mutations have been described to date (7 ), the most frequent ones being located in exon 10. Indeed, the M694V, V726A, M680I, and M694I mutations account for 65–95% of FMF alleles depending on the ethnic origin of the patient (8 ). E148Q is a frequent sequence variation situated in exon 2, but its involvement in the development of the disease remains controversial (9 ). The molecular diagnosis of FMF is based on various methods, including tedious and timeconsuming scanning techniques such as denaturing gradient gel electrophoresis (DGGE) (3 ) or direct sequencing (10 ). Restriction enzyme analysis enables the detection of known mutations but still requires multiple DNA amplifications. An in-house amplification refractory mutation system has been designed, but it detects only three mutations (11 ). Because the spectrum of the most frequent mutations in FMF has been characterized in all at-risk populations, a new method aimed at identifying a set of selected common mutations in a single step would be less time-consuming and would provide greater throughput than previous methods. We investigated the practicality and the reliability of the FMF StripAssay (ViennaLab Labordiagnostika), the first commercially available assay that allows the detection of 12 MEFV gene mutations in 1 working day. Twelve mutations located in exons 2 (E148Q), 3 (P369S), 5 (F479L),
