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INTERNATIONAL JOURNAL OF MOLECULAR MEDICINE 9: 91-94, 2002. Allelic variation of BAT-26 and BAT-40 poly-adenine repeat loci in North Indians.
INTERNATIONAL JOURNAL OF MOLECULAR MEDICINE 9: 91-94, 2002

Allelic variation of BAT-26 and BAT-40 poly-adenine repeat loci in North Indians MONISHA MUKHERJEE1'*, MINAL VAISH1'*, R.D. MITTAL2 and BALRAJ MITTAL1 Departments of Medical Genetics and Urology, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Raebareli Road, Lucknow 226014, India Received October 12, 2001; Accepted November 15, 2001

Abstract. Analysis of mononucleotide repeats BAT-26 and BAT-40 in North Indians revealed that there were germline polymorphisms at both the loci. We evaluated BAT-26 and BAT-40 in 100 normal healthy individuals from North India. The DNA from normal blood was PCR amplified using primers flanking the BAT-26 and BAT-40 loci. The allelic variation of BAT-26 and BAT-40 ranged between 117-130 and 94-112 bp respectively. The most frequent BAT-26 allele was 122 bp, which corresponded to 26 repeats and had a frequency of 32% while that of BAT-40 was 109 bp corresponding to 39 repeats with a frequency of 26%. These results suggest that poly­ morphisms in these poly-adenine repeat loci limit their 'quasimonomorphic' applicability in studying the microsatellite instability in cancers. Introduction The BAT-26 locus contains a 26-repeat adenine tract (poly A) and is located within the fifth intron of the MSH2 gene (1) while the BAT-40 is a poly (A) tract in the second intron of 3-ß-hydroxysteroid dehydrogenase gene (2). These mono­ nucleotide repeat markers are highly sensitive and specific indicators of generalized microsatellite instability as evident in human cancers (3,4). Many studies in different populations have shown that BAT-26 and BAT-40 exhibited very little variation not exceeding two nucleotides in germline DNA (5-9). This 'quasimonomorphic' nature had allowed their use in comparing the tumoral microsatellite instability with germline data rather than with the respective normal DNAs. However, germline polymorphisms in BAT-26 and BAT-40 were identified in African-Americans (3) and Japanese (10)

Correspondence to: Dr Balraj Mittal, Department of Medical Genetics, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Raibarelly Road, Lucknow 226014, India E-mail: balraj ©sgpgi.ac.in, [email protected] "Contributed equally Key words: mononucleotide repeat, microsatellite, MSH2 gene, 3ß-hydroxysteroid dehydrogenase gene

populations, which made it clear that accurate evaluation of the instability status of these microsatellites requires comparison of tumor results with the respective germline DNA. The hetero­ zygosity status at the poly-adenine loci was also studied in the Europeans, Americans and Japanese (6,10). Since allelic variations have been observed in different ethnic groups in various parts of the world, we have tried to define the allelic profiles, frequencies and heterozygosity at the twomicrosatellite loci, BAT-26 and BAT-40 in healthy individuals from North India. Materials and methods DNA isolation. Blood was collected in 0.5 M EDTA vials from normal healthy unrelated individuals of North India. DNA was isolated from whole blood according to the standard phenol-chloroform method (11). Mononucleotide repeat microsatellite analysis. The primers used to amplify microsatellite sequences at BAT-26 are 5'TGACTACTTTTGACTTCAGCC-3' and 5'-AACCATTCA ACATTTTTAACCC-3'; and for BAT-40 are 5'-ACAACC CTGCTTTTGTTCCT-3' and 5'-GTAGAGCAAGACCACTT G-3' (2). Polymerase chain reaction (PCR) amplification of DNA was performed with primers at 40 ng each, 200 p,M dNTPs, IX PCR buffer, 0.25 units of Taq polymerase (Genetix), 100 ng DNA and 0.2 ja.1 [a-32P]-CTP (10 \iC\l\u\ sp. act. 4000 Ci/mM) (BRIT, India) in a total volume of 26 \xl PCR conditions involved an initial denaturation at 95 °C for 3 min followed by 30 cycles (95°C for 1 min, 55°C for 2 min and 72°C for 3 min) and a final extension at 72°C for 8 min. All the PCR products were mixed with equal volume of formamide loading dye (95% formamide, 20 mM EDTA, 0.05% bromophenol blue, 0.05% xylene cyanol), denatured for 3 min at 95°C and loaded onto an 8% Polyacrylamide gel containing 7 M urea. Gels were run at 55 W for 2 h along with a DNA sequencing ladder. The gels were transferred onto a Whatman sheet, dried under vacuum and exposed to X-ray film (Kodak) for required time. Preparation of DNA ladder. For sizing different alleles of BAT-26 and BAT-40 a sequencing ladder of pUC18 plasmid DNA was prepared using a 5'-end universal labelled primer by di-deoxy thermosequenase cycle sequencing kit (USBAmersham Life Sciences, Cleveland, OH). The PCR product

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MUKHERJEE et al: BAT-26 AND BAT-40 ALLELIC VARIATION IN NORTH INDIANS

Figure l. Allelic profiles of mononucleotide repeat microsatellites in normal individuals. Lanes 1-11, BAT-26 alleles; A, T, G, C, DNA sequencing ladder; lanes 12-16, BAT-40 alleles.

Figure 3. Comparison of allele frequencies amongst different populations. The most frequent allele of the microsatellites is scored as 0. A, BAT-26 allelic profiles in North Indians, Americans and CEPH. The 122 bp PCR product corresponded to 26 repeats of adenine. B, BAT-40 allelic profiles in North Indians, Americans, CEPH and Japanese population. The 109 bp PCR product corresponded to 39 adenine repeats, which had maximum frequency in our population.

Results

Figure 2. Size variations of mononucleotide repeat microsatellite alleles in North Indians. A, frequency of BAT-26 PCR products. B, frequency of BAT-40 PCR products.

of 122 bp corresponded to 26-repeats in case of BAT-26 while a 109 bp product of BAT-40 corresponded to 39 repeats in BAT-40. The manual counting in order to determine the allele size may be liable to an error of ±1 bp.

Changes in the allelic profiles and frequency of the poly (A) mononucleotide repeats BAT-26 and BAT-40 were evaluated in 100 normal healthy individuals from North India. Fig. 1 shows the allelic pattern of the PCR products of BAT-26 and BAT-40 loci upon amplification from normal blood DNA. All the individuals were found to be homozygous for this allele. The allelic pattern of BAT-40 was also found to be homozygous. Fig. 2A depicts the frequency distribution of BAT-26 alleles in North Indians. The most prevalent BAT-26 allele (32%) was found to be a PCR product of 122 bp that corresponded to 26 repeats. The second most frequent allele was 121 bp (27%) followed by 124 bp (18%). The variation of BAT-26 was observed from -5 to +8 bp when compared to the major allele scored as 0. The frequency distribution of BAT-40 alleles is demonstrated in Fig. 2B. The most frequent one being 109 bp (26%) that corresponded to 39 adenine repeats. The second major allele was 108 bp (15%) followed by 106 (13%) and 104 bp (12%). BAT-40 showed a high range of variation from -15 to +3 bp. Allelic frequencies of BAT-26 and BAT-40 as observed in our study and those of Americans, families from Centre d'Etude pour le Polymorphisme Humain (CEPH) and Japanese are compared in Fig. 3A and B respectively.

INTERNATIONAL JOURNAL OF MOLECULAR MEDICINE 9: 91-94, 2002

Discussion Microsatellites are repeating units of 1-6 bp that are ubiquitous, abundant and highly polymorphic in eukaryotic genomes (12-14). Mutations within microsatellites are frequent, altering their overall length by insertion or deletion of a small number of repeat units with a rate as high as 1 0 3 in humans (15). D e s p i t e their high m u t a b i l i t y , stable allele frequency distributions are typically observed for microsatellites in humans as well as other primates (16,17). The degree of poly­ morphism of a microsatellite reflects two balancing factors: the frequency of strand slippage events during replication (18) and the efficiency of repair of resulting mismatches. Malfunction of the mismatch repair system results in a mutator phenotype, which is characterized by microsatellite instability (MSI). MSI is frequently detected in HNPCC spectrum cancers and in a subset of sporadic carcinomas (19). The mononucleotide microsatellites were believed to have quasimonomorphic allelic patterns where MSI in tumors could be determined on these loci without corresponding normal DNA (5-9). In subsequent studies this nature of the microsatellites was questioned and the germline polymorphisms at BAT-26 and BAT-40 loci limited their utility in determination of MSI without corresponding DNA, thereby supporting the need for thorough population studies to define the different allelic profiles and frequencies at microsatellite loci (4). The polymorphic nature of the microsatellite loci was confirmed in many ethnic groups like the African-Americans and Nigerians (4), CEPH family members (6) and Japanese populations (10). In our study on North Indians as well, a similar allelic variation was found with a size range of 117130 bp and 94-112 bp at BAT-26 and BAT-40 loci respectively. Amongst the two poly-adenine markers studied, a greater allelic variation was observed at the BAT-40 loci (-15 to +3 bp) as compared to BAT-26 (-5 to +8 bp). The distribution of poly (A) at BAT-26 locus in North Indians has been found to be comparable with that of the American, French and Japanese populations. In the present study, the North Indians also showed 122 bp as the major allele at BAT-26 locus, which corresponded to 26-repeats of adenine as observed in African-Americans. It was shown by Zhou et al (6) that 80% of individuals in a CEPH family showed the major allele with a variation ranging from -2 to + 1 bp. Samowitz et al (3) showed the major allele as 120 or more bp followed by 111, 112 and 114 bp alleles of BAT26. The frequency distribution of BAT-40 amongst the AfricanAmericans showed that 37 poly (A) repeat was present as the major allele in 61.6% of the population followed by the allele with 40 adenine repeats in 60.6% individuals (3). There was a wide variation of BAT-40 allele in African-Americans (-23 to +7 bp) as compared to the North Indians (-15 to +3 bp) and Japanese population (-17 to +5 bp). In contrast, the CEPH family members showed much less variation (-8 to +3 bp) at the BAT-40 locus (6) with a 4 8 % frequency of the major allele. The North Indians showed the 109 bp product as the most frequent BAT-40 allele which corresponded to 39 repeats while in the Japanese population the most frequent allele of BAT-40 was 126 bp (40 repeats) with a frequency of 49.51% followed by 18.69% at 39-adenine repeat locus (10).

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The allelic patterns of BAT-25 and BAT-26 in European and Japanese-Hawaiian populations appeared highly homo­ geneous with one major allele and small ranges of normal size variation for both loci (5-9). A small Nigerian population showed 2 5 % heterozygosity at 26 and 16-adenine repeats. The BAT-26 allele did not; however, show any heterozygosity in the North Indians. In contrast to the high percentage (72%) of B A T - 4 0 h e t e r o z y g o s i t y r e p o r t e d in E u r o p e a n s and Americans and moderate heterozygosity (14.6%) in the Japanese population, the North Indians showed no hetero­ zygosity. Due to the Taq polymerase stammering (6) during PCR reaction it was difficult to determine the closely spaced heterozygosity of the BAT-40 allele. The size of the major allele has been reported in the present study. The present work provides probably the first study of this nature from India. We believe that the study on BAT-26 and BAT-40 monoallelic variants in North Indians should provide useful background knowledge in the MSI determination which is an important aspect in cancer diagnosis and management. Acknowledgements The authors are grateful to the Director, Sanjay Gandhi PGIMS and Head of the Medical Genetics Department for providing the necessary facilities. Two of us (MM and MV) are thankful to Council of Scientific and Industrial Research, New Delhi, for awarding research fellowships. References 1. Liu B, Parsons RE, Hamilton SR, Petersen GM, Lynch HT, Watson P, Markovitz S, Willson JKV, Green J, de la Chapelle A, Kinzler KW and Vogelstein B: fiMSH2 mutations in hereditary nonpolyposis colorectal cancer kindreds. Cancer Res 54: 45904594, 1994. 2. Parsons R, Myeroff LL, Liu B, Willson JKV, Markovitz S, Kinzler KW and Vogelstein B: Microsatellite instability and mutations of the transforming growth factor ß type II receptor gene in colorectal cancer. Cancer Res 55: 5548-5550, 1995. 3. Samowitz WS, Slattery ML, Potter JD and Leppert MF: BAT-26 and BAT-40 instability in colorectal adenoma and carcinomas and germline polymorphisms. Am J Pathol 154: 1637-1641, 1999. 4. Pyatt R, Chadwick RB, Johnson CK, Adebamowo C, de la Chapelle A and Prior TW: Polymorphie variation at the BAT-25 and BAT-26 loci in individuals of African origin: implications for microsatellite instability testing. Am J Pathol 155: 349-353, 1999. 5. Hoang J-M, Cottu PH, Thuille B, Salmon RJ, Thomas G and Hamelin R: BAT-26, an indicator of the replication error pheno­ type in colorectal cancers and cell lines. Cancer Res 57: 300-303, 1997. 6. Zhou X-P, Hoang J-M, Cottu P, Thomas G and Hamelin R: Allelic profiles of mononucleotide repeat microsatellites in control individuals and in colorectal tumours with and without replication errors. Oncogene 15: 1713-1718, 1997. 7. Aaltonen LA, Salovaara R, Kristo P, Canzian F, Hemminki A, Petromaki P, Chadwick RB, Kaariainen H, Eskelinin M, Jarvinen H, Mecklin J-P and de la Chapelle A: Incidence of hereditary non-polyposis colorectal cancer and the feasibility of molecular screening for the disease. N Engl J Med 338: 1481-1487, 1998. 8. Grady WM, Rajput A, Myeroff L, Liu DF, Kwon K, Willis J and Markowitz S: Mutation of the type II transforming growth factor ß receptor is coincident with the transformation of human colon adenomas to malignant carcinomas. Cancer Res 58: 3101-3104, 1998. 9. Prior TW, Chadwick RB, Papp AC, Arcot AN, Isa AM, Pear OK, Stemmermann G, Percesepe A, Loukola A, Aaltonen LA and de la Chapelle A: The 11307K polymorphism of the APC gene in colorectal cancer. Gastroenterology 116: 1-7, 1999.

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