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Association of p53 codon 72 polymorphism with endometriosis
Association of p53 codon 72 polymorphism with endometriosis Maria Ammendola, M.D.,a Fulvia Gloria-Bottini, M.D.,b Francesco Sesti, M.D.,a Emilio Piccione, M.D.,a and Egidio Bottini, M.D.b a Division of Obstetrics and Gynecology, Department of Surgery, and b Division of Human Population Biopathology and Environmental Pathology, Department of Biopathology and Imaging Diagnostics, University of Rome ‘‘Tor Vergata,’’ School of Medicine, Rome, Italy
Objective: To study the association of endometriosis with p53 codon 72 polymorphism in the population of central Italy and to search for possible interaction with the PTPN22 polymorphism. Design: Study of p53 and PTPN22 polymorphisms in women with endometriosis. Analysis of PTPN22 genotype distribution in relation to p53 genotypes. Setting: Department of Obstetrics and Gynecology of the University of Rome ‘‘Tor Vergata.’’ Patient(s): The study included 129 women with endometriosis and 147 controls from the Caucasian population of central Italy. Intervention(s): None. Main Outcome Measure(s): Evaluation of risk for endometriosis. Result(s): No significant difference in the distribution of p53 codon 72 genotypes was observed between endometriosis patients and controls. An interaction between p53 and PTPN22 was observed: a protective action by the Arg/ Arg genotype against endometriosis seems to be present only in carriers of the *T allele of PTPN22. Conclusion(s): The negative association between the Arg/Arg genotype of p53 codon 72 found in Chinese people has not been observed in Japanese and Italian populations. Interaction with genes showing different allele frequencies among ethnic groups could be responsible for the differences reported among human populations concerning the relationship between p53 and susceptibility to endometriosis. (Fertil Steril 2008;90:406–8. 2008 by American Society for Reproductive Medicine.) Key Words: Endometriosis, p53 polymorphism, PTPN22 polymorphism
Endometriosis is a relatively benign disease that shows different prevalence among ethnic groups. The disease has a multifactorial origin with a polygenic component and presents a definite risk for malignant degeneration.
Here we report a study on the Caucasian population from central Italy.
In consideration of this risk, attention has been recently paid to a possible role of p53 genetic variants, in particular of codon 72. This codon shows a single nucleotide substitution resulting in the presence of either arginine or proline in the amino acidic sequence. The amino acid change affects biochemical and functional properties of p53: the proline variant is a stronger transcriptional activator, whereas the arginine variant is a stronger apoptosis inducer.
MATERIALS AND METHODS A total of 129 women with endometriosis were studied at the Department of Obstetrics and Gynecology of the University of Rome ‘‘Tor Vergata.’’ Indication for laparoscopy included chronic pelvic pain, infertility, ovarian cysts, and myomas. All women underwent complete presurgery clinical examination before the diagnostic operative laparoscopy. Endometriosis was always diagnosed by laparoscopic intervention, and criteria for inclusion in the study were those proposed by Holt and Weiss (5).
In an investigation in a Chinese population, Chang et al. (1) reported a negative association of the arginine variant with endometriosis, suggesting a protective action of the homozygous Arg/Arg genotype against the disease. The association has been confirmed in Chinese (2) but not in Japanese populations (3). In a recent study on the population of northern Italy, Lattuada et al. (4) did not find such an association. Received October 5, 2006; revised June 16, 2007; accepted June 18, 2007. Reprint requests: Fulvia Gloria-Bottini, M.D., Department of Biopathology and Imaging Diagnostics, University of Rome ‘‘Tor Vergata,’’ Via Montpellier 1, 00133 Rome, Italy (FAX: 39-0672596028; E-mail: gloria@med. uniroma2.it).
406
As controls a sample of 147 newborn infants from the same Caucasian population was studied. Blood was taken by the placental side of the umbilical vein after its section. Women gave verbal informed consent for participation of their newborn infants to this study, which was approved by the Department of Biopathology and Imaging Diagnostics. In consideration of the observational character of the study and of the fact that the participants are not identified, formal approval by the ethics committee was not necessary. The p53 polymorphism was evaluated using the restriction fragment length polymorphism polymerase chain reaction
Fertility and Sterility Vol. 90, No. 2, August 2008 Copyright ª2008 American Society for Reproductive Medicine, Published by Elsevier Inc.
0015-0282/08/$34.00 doi:10.1016/j.fertnstert.2007.06.051
TABLE 1 Distribution of p53 codon 72 polymorphism in endometriosis patients and controls from central Italy. Genotypesa
Endometriosis Controls a b
Allelesb
Arg/Arg
Arg/Pro
Pro/Pro
n
*Arg
*Pro
n
45.7% 46.3%
44.2% 40.1%
10.1% 13.6%
129 147
67.8% 66.3%
32.2% 33.7%
258 294
Endometriosis vs. controls: c2 (2 df) ¼ 0.987; P¼ .610. Endometriosis vs. controls: c2 (1 df) ¼ 0.081; P¼ .776.
Ammendola. p53 and endometriosis. Fertil Steril 2008.
method described by De La Calle-Martin et al. (6). The primer sequences corresponding to the fourth exon of the human p53 gene were as follows: sense oligo 50 -AATGG ATGATTTGATGCTGTCCC-30 and antisense oligo 50 -GG TGCAAGTCACAGACTTGGC-30 . Polymerase chain reactions were carried out in a total volume of 25 mL containing 200 ng of genomic DNA, 0.4 pmol of each primer, 2 mmol/L MgCl2, 200 mmol/L deoxynucleotide triphosphates, 1 buffer, and 2 U Taq polymerase. The amplification was performed for 35 cycles with an annealing temperature of 62 C. The amplified DNA was digested for 3 hours with 3 U of AccII restriction enzyme. The DNA fragments were resolved by electrophoresis through a 3% agarose gel. The c2 test of independence was performed with commercial software (SPSS, Chicago, IL).
RESULTS Table 1 shows the distribution of genotypes and alleles of p53 polymorphism in women with endometriosis and in controls from central Italy. No significant difference was observed between endometriosis patients and controls. Among controls, 74 were boys and 73 girls. No statistically significant differences were observed in the distribution of p53 polymorphism between sexes. Arg/Arg was 45.9% in boys and 46.6% in girls; Arg/Prol was 43.2% in boys and 37.0% in girls; Prol/ Prol was 10.8% in boys and 16.4% in girls. No significant association was found between p53 polymorphism and the following parameters: smoke, allergy, estrogen/progesterone treatment, dysmenorrhea, leiomyomas, pain, cyst, dimension of lesion, adhesion, surgical intervention, number of children, disturbances of menstrual cycle, relapse, stage, age, or body mass index. We recently reported an association between endometriosis and PTPN22 polymorphism (7). This prompted us to search for possible interactions between p53 and PTPN22 concerning their effect on susceptibility to endometriosis. Among *C/*C genotype of PTPN22, the frequency of carriers of the Prol allele of p53 is 50.9%, whereas among carriers of the *T allele of PTPN22 the frequency of the Prol allele of p53 is 72.7% (P¼0.06). These data suggest that in the presence of the *T allele of PTP22, carriers of the Pro alFertility and Sterility
lele are more susceptible to endometriosis as compared with Arg/Arg genotypes.
DISCUSSION The association between endometriosis and Arg72Prolp53 polymorphism found in Chinese people has not been found in Italians. It is possible that some of the female infants considered as controls have the genetic makeup to develop endometriosis in the future, thus reducing the identification of potential differences between groups. At present it has not been possible to study adult women without endometriosis as controls. It is interesting, however, that genotype frequencies in male infants are equal to those of women with endometriosis. The interaction with PTPN22 suggests a possible explanation for the differences observed among human populations. Interactions between genes are very common and may have an important role in determining the susceptibility to multifactorial disease. The overall protective role of Arg/Arg against endometriosis observed in the Chinese population seems also to be present in the Italian population but only in subjects carrying the *T allele of PTPN22 polymorphism. At present the genetic analysis of multifactorial disorders is a central problem in medical genetics, and there is a general consensus regarding the necessity of a non-reductionist approach. The analysis of single genetic factors, based on a Mendelian perspective, cannot solve the problem. It would be more productive to define a set of genes functionally related to the disease and to study simultaneously genetic and environmental factors involved in the susceptibility to the disease. A comprehensive approach will help to evaluate gene–gene and gene–environment interactions and to understand pathogenic mechanisms and differences between ethnic groups. REFERENCES 1. Chang CC, Hsieh YY, Tsai CH, Tsai HD, Lin CC. The proline form of P53 codon 72 polymorphism is associated with endometriosis. Fertil Steril 2002;77:43–5. 2. Hsieh YY, Lin CS. P53 codon 11,72 and 258 gene polymorphisms in endometriosis. Int J Biol Sci 2006;2:188–93.
407
3. Omori S, Yoshida S, Kennedy SH, Negro K, Hamana S, Barlow DH, Maruo T. Polymorphism at codon 72 of the p53 gene is not associated with endometriosis in a Japanese population. J Soc Gynecol Investig 2004;11:232–6. 4. Lattuada D, Vigan o P, Somiglianza E, Abbiati A, Candiani M, Di Blasio AM. Analysis of the codon 72 polymorphism of the TP53 gene in patients with endometriosis. Mol Hum Reprod 2004;10:651–4.
408
Ammendola et al.
p53 and endometriosis
5. Holt VL, Weiss NS. Recommendations for the design of epidemiologic studies of endometriosis. Epidemiology 2000;11:654–9. 6. De La Calle-Martin O, Fabregat V, Romero M, Soler J, Vives J, Yague I. AccII polymorphism of the p53 gene. Nucleic Acids Res 1990;18:4963. 7. Ammendola M, Bottini N, Pietropolli A, Saccucci P, Gloria-Bottini F. Association between PTPN22 and endometriosis. Fertil Steril. In press.
Vol. 90, No. 2, August 2008
Objective: To study the association of endometriosis with p53 codon 72 polymorphism in the population of central Italy and to search for possible interaction with the PTPN22 polymorphism. Design: Study of p53 and PTPN22 polymorphisms in women with endometriosis. Analysis of PTPN22 genotype distribution in relation to p53 genotypes. Setting: Department of Obstetrics and Gynecology of the University of Rome ‘‘Tor Vergata.’’ Patient(s): The study included 129 women with endometriosis and 147 controls from the Caucasian population of central Italy. Intervention(s): None. Main Outcome Measure(s): Evaluation of risk for endometriosis. Result(s): No significant difference in the distribution of p53 codon 72 genotypes was observed between endometriosis patients and controls. An interaction between p53 and PTPN22 was observed: a protective action by the Arg/ Arg genotype against endometriosis seems to be present only in carriers of the *T allele of PTPN22. Conclusion(s): The negative association between the Arg/Arg genotype of p53 codon 72 found in Chinese people has not been observed in Japanese and Italian populations. Interaction with genes showing different allele frequencies among ethnic groups could be responsible for the differences reported among human populations concerning the relationship between p53 and susceptibility to endometriosis. (Fertil Steril 2008;90:406–8. 2008 by American Society for Reproductive Medicine.) Key Words: Endometriosis, p53 polymorphism, PTPN22 polymorphism
Endometriosis is a relatively benign disease that shows different prevalence among ethnic groups. The disease has a multifactorial origin with a polygenic component and presents a definite risk for malignant degeneration.
Here we report a study on the Caucasian population from central Italy.
In consideration of this risk, attention has been recently paid to a possible role of p53 genetic variants, in particular of codon 72. This codon shows a single nucleotide substitution resulting in the presence of either arginine or proline in the amino acidic sequence. The amino acid change affects biochemical and functional properties of p53: the proline variant is a stronger transcriptional activator, whereas the arginine variant is a stronger apoptosis inducer.
MATERIALS AND METHODS A total of 129 women with endometriosis were studied at the Department of Obstetrics and Gynecology of the University of Rome ‘‘Tor Vergata.’’ Indication for laparoscopy included chronic pelvic pain, infertility, ovarian cysts, and myomas. All women underwent complete presurgery clinical examination before the diagnostic operative laparoscopy. Endometriosis was always diagnosed by laparoscopic intervention, and criteria for inclusion in the study were those proposed by Holt and Weiss (5).
In an investigation in a Chinese population, Chang et al. (1) reported a negative association of the arginine variant with endometriosis, suggesting a protective action of the homozygous Arg/Arg genotype against the disease. The association has been confirmed in Chinese (2) but not in Japanese populations (3). In a recent study on the population of northern Italy, Lattuada et al. (4) did not find such an association. Received October 5, 2006; revised June 16, 2007; accepted June 18, 2007. Reprint requests: Fulvia Gloria-Bottini, M.D., Department of Biopathology and Imaging Diagnostics, University of Rome ‘‘Tor Vergata,’’ Via Montpellier 1, 00133 Rome, Italy (FAX: 39-0672596028; E-mail: gloria@med. uniroma2.it).
406
As controls a sample of 147 newborn infants from the same Caucasian population was studied. Blood was taken by the placental side of the umbilical vein after its section. Women gave verbal informed consent for participation of their newborn infants to this study, which was approved by the Department of Biopathology and Imaging Diagnostics. In consideration of the observational character of the study and of the fact that the participants are not identified, formal approval by the ethics committee was not necessary. The p53 polymorphism was evaluated using the restriction fragment length polymorphism polymerase chain reaction
Fertility and Sterility Vol. 90, No. 2, August 2008 Copyright ª2008 American Society for Reproductive Medicine, Published by Elsevier Inc.
0015-0282/08/$34.00 doi:10.1016/j.fertnstert.2007.06.051
TABLE 1 Distribution of p53 codon 72 polymorphism in endometriosis patients and controls from central Italy. Genotypesa
Endometriosis Controls a b
Allelesb
Arg/Arg
Arg/Pro
Pro/Pro
n
*Arg
*Pro
n
45.7% 46.3%
44.2% 40.1%
10.1% 13.6%
129 147
67.8% 66.3%
32.2% 33.7%
258 294
Endometriosis vs. controls: c2 (2 df) ¼ 0.987; P¼ .610. Endometriosis vs. controls: c2 (1 df) ¼ 0.081; P¼ .776.
Ammendola. p53 and endometriosis. Fertil Steril 2008.
method described by De La Calle-Martin et al. (6). The primer sequences corresponding to the fourth exon of the human p53 gene were as follows: sense oligo 50 -AATGG ATGATTTGATGCTGTCCC-30 and antisense oligo 50 -GG TGCAAGTCACAGACTTGGC-30 . Polymerase chain reactions were carried out in a total volume of 25 mL containing 200 ng of genomic DNA, 0.4 pmol of each primer, 2 mmol/L MgCl2, 200 mmol/L deoxynucleotide triphosphates, 1 buffer, and 2 U Taq polymerase. The amplification was performed for 35 cycles with an annealing temperature of 62 C. The amplified DNA was digested for 3 hours with 3 U of AccII restriction enzyme. The DNA fragments were resolved by electrophoresis through a 3% agarose gel. The c2 test of independence was performed with commercial software (SPSS, Chicago, IL).
RESULTS Table 1 shows the distribution of genotypes and alleles of p53 polymorphism in women with endometriosis and in controls from central Italy. No significant difference was observed between endometriosis patients and controls. Among controls, 74 were boys and 73 girls. No statistically significant differences were observed in the distribution of p53 polymorphism between sexes. Arg/Arg was 45.9% in boys and 46.6% in girls; Arg/Prol was 43.2% in boys and 37.0% in girls; Prol/ Prol was 10.8% in boys and 16.4% in girls. No significant association was found between p53 polymorphism and the following parameters: smoke, allergy, estrogen/progesterone treatment, dysmenorrhea, leiomyomas, pain, cyst, dimension of lesion, adhesion, surgical intervention, number of children, disturbances of menstrual cycle, relapse, stage, age, or body mass index. We recently reported an association between endometriosis and PTPN22 polymorphism (7). This prompted us to search for possible interactions between p53 and PTPN22 concerning their effect on susceptibility to endometriosis. Among *C/*C genotype of PTPN22, the frequency of carriers of the Prol allele of p53 is 50.9%, whereas among carriers of the *T allele of PTPN22 the frequency of the Prol allele of p53 is 72.7% (P¼0.06). These data suggest that in the presence of the *T allele of PTP22, carriers of the Pro alFertility and Sterility
lele are more susceptible to endometriosis as compared with Arg/Arg genotypes.
DISCUSSION The association between endometriosis and Arg72Prolp53 polymorphism found in Chinese people has not been found in Italians. It is possible that some of the female infants considered as controls have the genetic makeup to develop endometriosis in the future, thus reducing the identification of potential differences between groups. At present it has not been possible to study adult women without endometriosis as controls. It is interesting, however, that genotype frequencies in male infants are equal to those of women with endometriosis. The interaction with PTPN22 suggests a possible explanation for the differences observed among human populations. Interactions between genes are very common and may have an important role in determining the susceptibility to multifactorial disease. The overall protective role of Arg/Arg against endometriosis observed in the Chinese population seems also to be present in the Italian population but only in subjects carrying the *T allele of PTPN22 polymorphism. At present the genetic analysis of multifactorial disorders is a central problem in medical genetics, and there is a general consensus regarding the necessity of a non-reductionist approach. The analysis of single genetic factors, based on a Mendelian perspective, cannot solve the problem. It would be more productive to define a set of genes functionally related to the disease and to study simultaneously genetic and environmental factors involved in the susceptibility to the disease. A comprehensive approach will help to evaluate gene–gene and gene–environment interactions and to understand pathogenic mechanisms and differences between ethnic groups. REFERENCES 1. Chang CC, Hsieh YY, Tsai CH, Tsai HD, Lin CC. The proline form of P53 codon 72 polymorphism is associated with endometriosis. Fertil Steril 2002;77:43–5. 2. Hsieh YY, Lin CS. P53 codon 11,72 and 258 gene polymorphisms in endometriosis. Int J Biol Sci 2006;2:188–93.
407
3. Omori S, Yoshida S, Kennedy SH, Negro K, Hamana S, Barlow DH, Maruo T. Polymorphism at codon 72 of the p53 gene is not associated with endometriosis in a Japanese population. J Soc Gynecol Investig 2004;11:232–6. 4. Lattuada D, Vigan o P, Somiglianza E, Abbiati A, Candiani M, Di Blasio AM. Analysis of the codon 72 polymorphism of the TP53 gene in patients with endometriosis. Mol Hum Reprod 2004;10:651–4.
408
Ammendola et al.
p53 and endometriosis
5. Holt VL, Weiss NS. Recommendations for the design of epidemiologic studies of endometriosis. Epidemiology 2000;11:654–9. 6. De La Calle-Martin O, Fabregat V, Romero M, Soler J, Vives J, Yague I. AccII polymorphism of the p53 gene. Nucleic Acids Res 1990;18:4963. 7. Ammendola M, Bottini N, Pietropolli A, Saccucci P, Gloria-Bottini F. Association between PTPN22 and endometriosis. Fertil Steril. In press.
Vol. 90, No. 2, August 2008