- Email: [email protected]
Genotyping by “Cold Single-Strand Conformation Polymorphism” of the UGT1A1 Promoter Polymorphism in Mexican Mestizos
Blood Cells, Molecules, and Diseases (2002) 28(1) Jan/Feb: 86 –90 doi:10.1006/bcmd.2001.0481, available online at http://www.idealibrary.com on
Ara´mbula and Vaca
Genotyping by “Cold Single-Strand Conformation Polymorphism” of the UGT1A1 Promoter Polymorphism in Mexican Mestizos Submitted 12/28/01 (Communicated by E. Beutler, M.D., 12/31/01)
Eliakym Ara´mbula1 and Gerardo Vaca1 ABSTRACT: Since no data have previously been reported concerning both the (TA)n polymorphism at the promoter of the UGT1A1 gene in the Mexican population and the use of single-strand conformation polymorphism (SSCP) for the detection of such polymorphism, genotyping by SSCP in 375 G-6-PD normal (Group A) and 81 G-6-PD-deficient (Group B) mestizos belonging to 14 states was carried out. Allele frequencies for (TA)6 and (TA)7 repeats were 0.654 and 0.334, respectively, in Group A and 0.685 and 0.315 in Group B; in the former group, the (TA)5 allele was also observed with a frequency of 0.012. The frequencies of the genotype (TA)7/(TA)7 were 10.1% (Group A) and 8.6% (Group B). The (TA)7/(TA)8 genotype was also observed in a patient with unconjugated hyperbilirubinemia. Due to the importance of its potential medical implications, the observed high frequency (10%) of the (TA)7/(TA)7 genotype is stressed. Genotyping by SSCP of the (TA)n polymorphism is an adequate methodological option. © 2002 Elsevier Science (USA) Key Words: polymorphism; UGT1A1; Gilbert syndrome; SSCP; Mexican mestizos.
INTRODUCTION
the Gilbert syndrome; total or partial UGT1A1 deficiency, as a result of mutations in any of the five exons of the UGT1A1 gene, in patients with the two former syndromes respectively have been demonstrated. By the other hand almost all the Caucasian individuals with Gilbert syndrome, a very common inborn error of bilirubin metabolism, are homozygous for the (TA)7 allele at the promoter of the UGT1A1 gene (1). Several methods for the detection of the (TA)n polymorphism have been described. These methods include (a) direct sequencing (2), (b) radioactive PCR followed by separation of the amplified products on 6% denaturing polyacrylamide gels and autoradiography (6), (c) denaturing high performance liquid chromatography (7), (d) melting temperature (Tm) analysis (8), (e) double gradient denaturing gradient gel electrophoresis (9), and (f) PCR and polyacrylamide gel electrophoresis followed by staining with either silver nitrate or ethidium bromide (10). However, procedures (a)
The enzyme UDP-glucuronosyltransferase (UGT1A1), encoded by the UGT1A1 gene, catalyzes the conjugation of bilirubin with glucuronic acid in liver. The UGT1A1 gene consists of five exons; exon 1 is specific for the UGT1A1 enzyme whereas exons 2–5 are common to all the members of the UGT1 locus located on chromosome 2q37 (1). The normal promoter of the UGT1A1 gene has a motif A(TA)6TAA with six TA repeats; another three alleles with five, seven, and eight TA repeats have been identified (2, 3). In several studies significant differences in the distribution of the four alleles in different ethnic groups have been observed (3–5). The promoters with (TA)7 or (TA)8 repeats exhibit a reduction in the transcription of the gene which in turn leads to a reduction in the activity of the UGT1A1 enzyme (2, 3). The inherited unconjugated hyperbilirubinemias include the Crigler–Najjar syndromes I and II and
Correspondence and reprint request to: Gerardo Vaca, Divisio´n de Gene´tica, Centro de Investigacio´n Biome´dica de Occidente (IMSS), Apartado Postal 2-1079, Guadalajara 2, Jalisco, CP 44281, Me´xico. Fax: (33) 36-72-80-71. E-mail: [email protected]. 1 Divisio´n de Gene´tica, Centro de Investigacio´n Biome´dica de Occidente, Instituto Mexicano del Seguro Social (IMSS), Guadalajara, Jalisco, Me´xico. 1079-9796/02 $35.00 © 2002 Elsevier Science (USA)
All rights reserved.
86
Ara´mbula and Vaca
Blood Cells, Molecules, and Diseases (2002) 28(1) Jan/Feb: 86 –90 doi:10.1006/bcmd.2001.0481, available online at http://www.idealibrary.com on
from G-6-PD-deficient individuals (Group B), both previously characterized for G6PD status at biochemical and molecular levels, have been retrospectively genotyped for the (TA)n polymorphism. Approximately 75% of the G-6-PD-deficient individuals showed some of the genotypes that define to the G-6-PD A- variant; partial results of the G-6-PD genotyping have been reported (13). The individuals from both groups are native to 14 different states of the Mexican Republic, mostly from Pacific, Gulf, and Northeastern states. DNA Analysis
MATERIALS AND METHODS
PCR conditions and primers were according to Kaplan et al. (10) and Sampietro et al. (14) respectively; these primers produce fragments of 73 bp for the (TA)6 allele. PCR products were subjected to electrophoresis on a 12% polyacrylamide gel (19:1) and then stained with silver nitrate (14). The SSCP procedure was basically performed according to Hongyo et al. (15). In brief, the composition of the reaction mixture for SSCP was 5.0 l of PCR product, 12.6 l of 1.25⫻ TBE buffer, 0.4 l of methyl mercury hydroxide, 2.0 l of 15% (w/w) Ficoll (MW 400,000) containing 0.1% (w/w) bromophenol blue and 0.1% (w/w) xylene cyanol. The mixture was denatured by heating at 95°C for 4 min followed by rapid cooling at 0°C with ice. The entire 20 l of each sample was applied to a nondenaturing 20% polyacrylamide gel (8 ⫻ 8 cm; acrylamide:bis ratio 37.5:1) with 5% glycerol. Electrophoresis was performed at 300 V constant for approximately 3 h at 15°C (temperature of the 1.25⫻ TBE running buffer) in an electrophoretic chamber connected to an external thermostatically controlled circulating water bath. The gels were stained with silver nitrate. Expected genotype frequencies were calculated from respective single allele frequencies according to the Hardy–Weinberg equation.
Subjects
RESULTS AND DISCUSSION
To date, 375 DNA samples from G-6-PD normal male adults (Group A) and 81 DNA samples
The observed genotypes determined by electrophoresis were 5-6, 5-7, 6-6, 6-7, and 7-7 (Fig.
FIG. 1. Genotyping by electrophoresis of the (TA)n polymorphism in the promoter of the UGT1A1 gene. Genotype assignment: 6-6, homozygous for (TA)6 allele; 7-7, homozygous for (TA)7 allele; 5-6, 6-7, and 7-8, heterozygous for the respective alleles.
to (d) are not readily available in many laboratories. Non-radioactive single strand conformation polymorphism (SSCP) could be a more accessible methodological option; we often use this procedure in order to detect mutations and polymorphisms at the G-6-PD gene. A project aiming to determine the molecular basis of G-6-PD deficiency in Mexico as well as the genotype at the promoter of the UGT1A1 gene in both G-6-PD-deficient and G-6-PD normal individuals is in progress in our laboratory (11–13). No data have previously been reported concerning the (TA)n polymorphism in the Mexican population. In this paper we present results of genotyping by SSCP of the promoter at the UGT1A1 gene in healthy G-6-PD normal as well as in G-6-PDdeficient Mexican mestizos.
87
Blood Cells, Molecules, and Diseases (2002) 28(1) Jan/Feb: 86 –90 doi:10.1006/bcmd.2001.0481, available online at http://www.idealibrary.com on
Ara´mbula and Vaca
TABLE 2 UGT1A1 Promoter Gene Frequencies (Number of Chromosomes) in 456 Mexican Mestizos Allele Group
5
6
7
A (n ⫽ 750 chr) B (n ⫽ 162 chr)
0.012 (8) 0
0.654 (491) 0.685 (111)
0.334 (251) 0.315 (51)
P ⫽ NS.
with a frequency of 0.012. The frequency of the genotype (TA)7/(TA)7 was 10.1% (Group A) and 8.6% (Group B) (P ⫽ NS), such frequency is similar to those reported in several populations of European origin (2, 3, 6). Genotype and allele frequencies were similar in both groups (P ⫽ NS) and within each group, the genotype frequencies were in Hardy–Weinberg equilibrium. We also observed the (TA)7/(TA)8 genotype in a patient with moderate unconjugated hyperbilirubinemia (total bilirubin 2.5 mg/dl, indirect 2.3 mg/dl; Hb ⫽ 15.3 g/dl; hematocrit ⫽ 46.3; reticulocytes 3.1%) who was referred to our laboratory in order to screening for inborn errors of red cell metabolism; the screening results were negative. The presence in the Mexican population of the (TA)5 and (TA)8 alleles, which apparently are specific for African populations (3, 4), can be explained by racial admixture. We have also observed another African polymorphisms at the G-6-PD gene in Mexican mestizos (13). Owing to the importance of its potential medical implications, the observed high frequency (10%) of the (TA)7/(TA)7 genotype is stressed. Thus, the genotype at the UGT1A1 gene promoter is an important determinant not only of the bilirubin levels in patients with diverse inherited hematologic disorders (10, 16 –18), newborns with prolonged unexplained jaundice (19) and ABOincompatible neonates (20) but also of the adverse effects of certain anticancer drugs in whose metabolism participates the enzyme UGT1A1 (21). Furthermore, an association between the UGT1A1 promoter polymorphism and an increased breast cancer risk in premenopausal women has been reported (22). Finally, genotyping by SSCP of the (TA)n polymorphism as described in this paper is an adequate methodological option.
FIG. 2. Genotyping by SSCP of the (TA)n polymorphism in the promoter of the UGT1A1 gene. Genotype assignment: 6-6, homozygous for (TA)6 allele; 7-7, homozygous for (TA)7 allele; 5-6, 5-7, 6-7, and 7-8, heterozygous for the respective alleles.
1). The determination of the correspondent SSCP patterns (Fig. 2) permits an unambiguous genotype assignment, particularly the heterozygous genotypes; mixtures (1:1) of PCR products with (TA)6/(TA)6 and (TA)7/(TA)7 genotypes, assigned by electrophoresis, given SSCP patterns equals to those produced by PCR-products with the heterozygous (TA)6/(TA)7 genotype. In a very few samples the genotypes assigned by both procedures were discordant; in these samples the definitive genotype assignment was by SSCP. Genotype and allele frequencies are shown in Tables 1 and 2, respectively. Allele frequencies for (TA)6 and (TA)7 repeats were 0.654 and 0.334, respectively, in Group A and 0.685 and 0.315 in Group B; in the former group the (TA)5 allele was also observed TABLE 1 UGT1A1 Promoter Genotypes in 456 Mexican Mestizos Genotype (%) Group
6-6
6-7
7-7
6-5
7-5
A (n ⫽ 375) B (n ⫽ 81)
41.3 45.7
46.4 45.7
10.1 8.6
1.9 0
0.3 0
P ⫽ NS.
88
Ara´mbula and Vaca
Blood Cells, Molecules, and Diseases (2002) 28(1) Jan/Feb: 86 –90 doi:10.1006/bcmd.2001.0481, available online at http://www.idealibrary.com on
ACKNOWLEDGMENTS 9.
We thank QFB’s Graciela Vega, Delia Pe´rez, and Marı´a Eugenia Medina and Drs. Jose´ Manuel Hadad Bello, Javier Gutie´rrez Almanza, Beatriz Rangel Guzma´n, Karina Silva, Jorge Pe´rez Osorio, and Rafael Miranda, Heads of the Blood Banks from the Regional Hospitals (IMSS) at Culiaca´n (Sin), Tepic (Nay), Colima (Col), Acapulco (Gro), Torreo´n (Coah), Ciudad Madero (Tams), Me´rida (Yuc), and San Luis Potosı´ (SLP), for providing blood samples.
10.
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Chowdhury, J. R., Wolkoff, A. W., Chowdhury, N. R., and Arias, I. W. (2001) Hereditary jaundice and disorders of bilirubin metabolism. In The Metabolic and Molecular Bases of Inherited Disease (Scriver, C. R., Beaudet, A. L., Valle, D., and Sly, W., Eds.), pp. 3063–3101. McGraw-Hill, New York. Bosma, P. J., Jayanta, R. C., Bakker, C., Gantla, S., De Boer, A., Oostra, B. A., Lindhout, D., Tytgat, G. N. J., Jansen, P. L. M., Oude Elferink, R. P. J., and Roy Chowdhury, N. (1995) The genetic basis of the reduced expression of bilirubin UDP-glucuronosyltransferase 1 in Gilbert’s syndrome. New Engl. J. Med. 333, 1171–1175. Beutler, E., Gelbart, T., and Demina, A. (1998) Racial variability in the UDP-glucuronosyltransferase 1 (UGT1A1) promoter: A balanced polymorphism for regulation of bilirubin metabolism. Proc. Natl. Acad. Sci. USA 95, 8170 – 8174. Hall, D., Ybazeta, G., Destro-Bisol, G., Petzl-Erler, M. L., and Di Rienzo, A. (1999) Variability at the uridine diphosphate glucuronosyltransferase 1A1 promoter in human populations and primates. Pharmacogenetics 9, 591–599. Lampe, W. J., Bigler, J., Horner, N. K., and Potter, J. D. (1999) UDP-glucuronosyltransferase (UGT1A1*28 and UGT1A6*2) polymorphisms in Caucasians and Asians: Relationships to serum bilirubin concentrations. Pharmacogenetics 9, 341–349. Monaghan, G., Ryan, M., Seddon, R., Hume, R., and Burchell, B. (1996) Genetic variation in bilirubin UDP-glucuronosyltransferase gene promoter and Gilbert’s syndrome. Lancet 347, 578 –581. Pirulli, D., Giordano, M., Puzzer, D., Crovella, S., Rigato, I., Tiribelli, C., Momigliano-Richiardi, P., and Amoroso, A. (2000) Rapid method for detection of extra (TA) in the promoter of the bilirubin-UDPglucuronosyltransferase 1 gene associated with Gilbert syndrome. Clin. Chem. 46, 129 –131. Marziliano, N., Pelo, E., Minuti, B., Passerini, I., Torricelli, F., and Da Prato, L. (2000) Melting tem-
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perature assay for a UGT1A gene variant in Gilbert syndrome. Clin. Chem. 46, 423– 425. Gu¨rtler, V., Parkin, J. D., and Mayall, B. C. (1999) Use of double gradient denaturing gradient gel electrophoresis to detect (AT)n polymorphisms in the UDP-glucuronosyltransferase 1 gene promoter associated with Gilbert’s syndrome. Electrophoresis 20, 2841–2843. Kaplan, M., Renbaum, P., Levy-Lahad, E., Hammerman, C., Lahad, A., and Beutler, E. (1997) Gilbert syndrome and glucose-6-phosphate dehydrogenase deficiency: A dose-dependent genetic interaction crucial to neonatal hyperbilirubinemia. Proc. Natl. Acad. Sci. USA 94, 12128 –12132. Vaca, G. (2001) Heterogeneidad molecular de la deficiencia de G-6-PD en Me´xico. XXVI Congreso Nacional de Gene´tica Humana. Puerto Vallarta, Jal. 23–26 de Octubre, p. 11. Libro de Resu´menes. Vaca, G., Ara´mbula, E., Vega, M. G., Pe´rez, D., and Medina, M. E. (1999) Genotyping of the glucose-6phosphate dehydrogenase (G6PD) gene and the promoter of the bilirubin UDP-glucuronosyltransferase 1 (UGT1A1) gene in Mexican mestizos. Eur. J. Hum. Genet. 7(Suppl. 1), 149. [abstract] Ara´mbula, E., Aguilar, J. C., and Vaca, G. (2000) Glucose-6-phosphate dehydrogenase mutations and haplotypes in Mexican mestizos. Blood Cells Mol. Dis. 26, 387–394. Sampietro, M., Lupica, L., Perrero, L., Comino, A., Martinez di Montemuros, F., Capellini, M. D., and Fiorelli, G. (1997) The expression of uridine diphosphate glucuronosyltransferase gene is a major determinant of bilirubin level in heterozygous -thalassaemia and in glucose-6-phosphate dehydrogenase deficiency. Br. J. Haematol. 99, 437– 439. Hongyo, T., Buzard, G. S., Calvert, R. J., and Weghorst, C. M. (1993) ‘Cold SSCP’: A simple, rapid and non-radioactive method for optimized single-strand conformation polymorphism analyses. Nucleic Acids Res. 21, 3637–3642. Galanello, R., Perseu, L., Melis, M. A., Cipollina, L., Barella, S., Giagu, N., Turco, M. P., Maccioni, O., and Cao, A. (1997) Hyperbilirubinaemia in heterozygous -thalassaemia is related to co-inherited Gilbert’s syndrome. Br. J. Haematol. 99, 433– 436. Iolascon, A., Faienza, M. F., Moretti, A., Perrotta, S., and Miraglia del Giudice, E. (1998) UGT1 promoter polymorphism accounts for increased neonatal appearance of hereditary spherocytosis. Blood 91, 1093. Passon, R. G., Howard, T. A., Zimmerman, S. A., Schultz, W. H., and Ware, R. E. (2001) Influence of bilirubin uridine diphosphate-glucuronosyltransferase 1A promoter polymorphism on serum bilirubin levels and cholelithiasis in children with sickle cell anemia. Am. J. Pediatr. Hematol. Oncol. 23, 448 – 451.
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Monaghan, G., McLellan, A., McGeehan, A., Li Volti, S., Mollica, F., Salemi, I., Din, Z., Cassidy, A., Hume, R., and Burchell, B. (1999) Gilbert’s syndrome is a contributory factor in prolonged unconjugated hyperbilirubinemia of the newborn. J. Pediatr. 134, 441– 446. Kaplan, M., Hammerman, C., Renbaum, P., Klein, G., and Levy-Lahad, E. (2000) Gilbert’s syndrome and hyperbilirubinemia in ABO-incompatible-neonates. Lancet 356, 652– 653. Iyer, L., Hall, D., Das, S., Ramı´rez, J., Mortell, M. A.,
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Kim, S., Di Rienzo, A., and Ratain, M. J. (1999) Phenotype/genotype correlation of in vitro SN-38 and bilirubin glucuronidation in human liver tissue with UGT1A1 promoter polymorphism. Clin. Pharmacol. Ther. 65, 576 –582. Guillemette, C., Millikan, R. C., Newman, B., and Housman, D. E. (2001) Genetic polymorphisms in uridine diphospho-glucuronosyltransferase 1A1 and association with breast cancer among African– Americans. Cancer Res. 60, 950 –956.
Ara´mbula and Vaca
Genotyping by “Cold Single-Strand Conformation Polymorphism” of the UGT1A1 Promoter Polymorphism in Mexican Mestizos Submitted 12/28/01 (Communicated by E. Beutler, M.D., 12/31/01)
Eliakym Ara´mbula1 and Gerardo Vaca1 ABSTRACT: Since no data have previously been reported concerning both the (TA)n polymorphism at the promoter of the UGT1A1 gene in the Mexican population and the use of single-strand conformation polymorphism (SSCP) for the detection of such polymorphism, genotyping by SSCP in 375 G-6-PD normal (Group A) and 81 G-6-PD-deficient (Group B) mestizos belonging to 14 states was carried out. Allele frequencies for (TA)6 and (TA)7 repeats were 0.654 and 0.334, respectively, in Group A and 0.685 and 0.315 in Group B; in the former group, the (TA)5 allele was also observed with a frequency of 0.012. The frequencies of the genotype (TA)7/(TA)7 were 10.1% (Group A) and 8.6% (Group B). The (TA)7/(TA)8 genotype was also observed in a patient with unconjugated hyperbilirubinemia. Due to the importance of its potential medical implications, the observed high frequency (10%) of the (TA)7/(TA)7 genotype is stressed. Genotyping by SSCP of the (TA)n polymorphism is an adequate methodological option. © 2002 Elsevier Science (USA) Key Words: polymorphism; UGT1A1; Gilbert syndrome; SSCP; Mexican mestizos.
INTRODUCTION
the Gilbert syndrome; total or partial UGT1A1 deficiency, as a result of mutations in any of the five exons of the UGT1A1 gene, in patients with the two former syndromes respectively have been demonstrated. By the other hand almost all the Caucasian individuals with Gilbert syndrome, a very common inborn error of bilirubin metabolism, are homozygous for the (TA)7 allele at the promoter of the UGT1A1 gene (1). Several methods for the detection of the (TA)n polymorphism have been described. These methods include (a) direct sequencing (2), (b) radioactive PCR followed by separation of the amplified products on 6% denaturing polyacrylamide gels and autoradiography (6), (c) denaturing high performance liquid chromatography (7), (d) melting temperature (Tm) analysis (8), (e) double gradient denaturing gradient gel electrophoresis (9), and (f) PCR and polyacrylamide gel electrophoresis followed by staining with either silver nitrate or ethidium bromide (10). However, procedures (a)
The enzyme UDP-glucuronosyltransferase (UGT1A1), encoded by the UGT1A1 gene, catalyzes the conjugation of bilirubin with glucuronic acid in liver. The UGT1A1 gene consists of five exons; exon 1 is specific for the UGT1A1 enzyme whereas exons 2–5 are common to all the members of the UGT1 locus located on chromosome 2q37 (1). The normal promoter of the UGT1A1 gene has a motif A(TA)6TAA with six TA repeats; another three alleles with five, seven, and eight TA repeats have been identified (2, 3). In several studies significant differences in the distribution of the four alleles in different ethnic groups have been observed (3–5). The promoters with (TA)7 or (TA)8 repeats exhibit a reduction in the transcription of the gene which in turn leads to a reduction in the activity of the UGT1A1 enzyme (2, 3). The inherited unconjugated hyperbilirubinemias include the Crigler–Najjar syndromes I and II and
Correspondence and reprint request to: Gerardo Vaca, Divisio´n de Gene´tica, Centro de Investigacio´n Biome´dica de Occidente (IMSS), Apartado Postal 2-1079, Guadalajara 2, Jalisco, CP 44281, Me´xico. Fax: (33) 36-72-80-71. E-mail: [email protected]. 1 Divisio´n de Gene´tica, Centro de Investigacio´n Biome´dica de Occidente, Instituto Mexicano del Seguro Social (IMSS), Guadalajara, Jalisco, Me´xico. 1079-9796/02 $35.00 © 2002 Elsevier Science (USA)
All rights reserved.
86
Ara´mbula and Vaca
Blood Cells, Molecules, and Diseases (2002) 28(1) Jan/Feb: 86 –90 doi:10.1006/bcmd.2001.0481, available online at http://www.idealibrary.com on
from G-6-PD-deficient individuals (Group B), both previously characterized for G6PD status at biochemical and molecular levels, have been retrospectively genotyped for the (TA)n polymorphism. Approximately 75% of the G-6-PD-deficient individuals showed some of the genotypes that define to the G-6-PD A- variant; partial results of the G-6-PD genotyping have been reported (13). The individuals from both groups are native to 14 different states of the Mexican Republic, mostly from Pacific, Gulf, and Northeastern states. DNA Analysis
MATERIALS AND METHODS
PCR conditions and primers were according to Kaplan et al. (10) and Sampietro et al. (14) respectively; these primers produce fragments of 73 bp for the (TA)6 allele. PCR products were subjected to electrophoresis on a 12% polyacrylamide gel (19:1) and then stained with silver nitrate (14). The SSCP procedure was basically performed according to Hongyo et al. (15). In brief, the composition of the reaction mixture for SSCP was 5.0 l of PCR product, 12.6 l of 1.25⫻ TBE buffer, 0.4 l of methyl mercury hydroxide, 2.0 l of 15% (w/w) Ficoll (MW 400,000) containing 0.1% (w/w) bromophenol blue and 0.1% (w/w) xylene cyanol. The mixture was denatured by heating at 95°C for 4 min followed by rapid cooling at 0°C with ice. The entire 20 l of each sample was applied to a nondenaturing 20% polyacrylamide gel (8 ⫻ 8 cm; acrylamide:bis ratio 37.5:1) with 5% glycerol. Electrophoresis was performed at 300 V constant for approximately 3 h at 15°C (temperature of the 1.25⫻ TBE running buffer) in an electrophoretic chamber connected to an external thermostatically controlled circulating water bath. The gels were stained with silver nitrate. Expected genotype frequencies were calculated from respective single allele frequencies according to the Hardy–Weinberg equation.
Subjects
RESULTS AND DISCUSSION
To date, 375 DNA samples from G-6-PD normal male adults (Group A) and 81 DNA samples
The observed genotypes determined by electrophoresis were 5-6, 5-7, 6-6, 6-7, and 7-7 (Fig.
FIG. 1. Genotyping by electrophoresis of the (TA)n polymorphism in the promoter of the UGT1A1 gene. Genotype assignment: 6-6, homozygous for (TA)6 allele; 7-7, homozygous for (TA)7 allele; 5-6, 6-7, and 7-8, heterozygous for the respective alleles.
to (d) are not readily available in many laboratories. Non-radioactive single strand conformation polymorphism (SSCP) could be a more accessible methodological option; we often use this procedure in order to detect mutations and polymorphisms at the G-6-PD gene. A project aiming to determine the molecular basis of G-6-PD deficiency in Mexico as well as the genotype at the promoter of the UGT1A1 gene in both G-6-PD-deficient and G-6-PD normal individuals is in progress in our laboratory (11–13). No data have previously been reported concerning the (TA)n polymorphism in the Mexican population. In this paper we present results of genotyping by SSCP of the promoter at the UGT1A1 gene in healthy G-6-PD normal as well as in G-6-PDdeficient Mexican mestizos.
87
Blood Cells, Molecules, and Diseases (2002) 28(1) Jan/Feb: 86 –90 doi:10.1006/bcmd.2001.0481, available online at http://www.idealibrary.com on
Ara´mbula and Vaca
TABLE 2 UGT1A1 Promoter Gene Frequencies (Number of Chromosomes) in 456 Mexican Mestizos Allele Group
5
6
7
A (n ⫽ 750 chr) B (n ⫽ 162 chr)
0.012 (8) 0
0.654 (491) 0.685 (111)
0.334 (251) 0.315 (51)
P ⫽ NS.
with a frequency of 0.012. The frequency of the genotype (TA)7/(TA)7 was 10.1% (Group A) and 8.6% (Group B) (P ⫽ NS), such frequency is similar to those reported in several populations of European origin (2, 3, 6). Genotype and allele frequencies were similar in both groups (P ⫽ NS) and within each group, the genotype frequencies were in Hardy–Weinberg equilibrium. We also observed the (TA)7/(TA)8 genotype in a patient with moderate unconjugated hyperbilirubinemia (total bilirubin 2.5 mg/dl, indirect 2.3 mg/dl; Hb ⫽ 15.3 g/dl; hematocrit ⫽ 46.3; reticulocytes 3.1%) who was referred to our laboratory in order to screening for inborn errors of red cell metabolism; the screening results were negative. The presence in the Mexican population of the (TA)5 and (TA)8 alleles, which apparently are specific for African populations (3, 4), can be explained by racial admixture. We have also observed another African polymorphisms at the G-6-PD gene in Mexican mestizos (13). Owing to the importance of its potential medical implications, the observed high frequency (10%) of the (TA)7/(TA)7 genotype is stressed. Thus, the genotype at the UGT1A1 gene promoter is an important determinant not only of the bilirubin levels in patients with diverse inherited hematologic disorders (10, 16 –18), newborns with prolonged unexplained jaundice (19) and ABOincompatible neonates (20) but also of the adverse effects of certain anticancer drugs in whose metabolism participates the enzyme UGT1A1 (21). Furthermore, an association between the UGT1A1 promoter polymorphism and an increased breast cancer risk in premenopausal women has been reported (22). Finally, genotyping by SSCP of the (TA)n polymorphism as described in this paper is an adequate methodological option.
FIG. 2. Genotyping by SSCP of the (TA)n polymorphism in the promoter of the UGT1A1 gene. Genotype assignment: 6-6, homozygous for (TA)6 allele; 7-7, homozygous for (TA)7 allele; 5-6, 5-7, 6-7, and 7-8, heterozygous for the respective alleles.
1). The determination of the correspondent SSCP patterns (Fig. 2) permits an unambiguous genotype assignment, particularly the heterozygous genotypes; mixtures (1:1) of PCR products with (TA)6/(TA)6 and (TA)7/(TA)7 genotypes, assigned by electrophoresis, given SSCP patterns equals to those produced by PCR-products with the heterozygous (TA)6/(TA)7 genotype. In a very few samples the genotypes assigned by both procedures were discordant; in these samples the definitive genotype assignment was by SSCP. Genotype and allele frequencies are shown in Tables 1 and 2, respectively. Allele frequencies for (TA)6 and (TA)7 repeats were 0.654 and 0.334, respectively, in Group A and 0.685 and 0.315 in Group B; in the former group the (TA)5 allele was also observed TABLE 1 UGT1A1 Promoter Genotypes in 456 Mexican Mestizos Genotype (%) Group
6-6
6-7
7-7
6-5
7-5
A (n ⫽ 375) B (n ⫽ 81)
41.3 45.7
46.4 45.7
10.1 8.6
1.9 0
0.3 0
P ⫽ NS.
88
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Blood Cells, Molecules, and Diseases (2002) 28(1) Jan/Feb: 86 –90 doi:10.1006/bcmd.2001.0481, available online at http://www.idealibrary.com on
ACKNOWLEDGMENTS 9.
We thank QFB’s Graciela Vega, Delia Pe´rez, and Marı´a Eugenia Medina and Drs. Jose´ Manuel Hadad Bello, Javier Gutie´rrez Almanza, Beatriz Rangel Guzma´n, Karina Silva, Jorge Pe´rez Osorio, and Rafael Miranda, Heads of the Blood Banks from the Regional Hospitals (IMSS) at Culiaca´n (Sin), Tepic (Nay), Colima (Col), Acapulco (Gro), Torreo´n (Coah), Ciudad Madero (Tams), Me´rida (Yuc), and San Luis Potosı´ (SLP), for providing blood samples.
10.
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