NS22: A Highly Polymorphic Complex MicrosatelliteMarker Within the ATM Gene

Nitin Udar,1,2 Shahriar Farzad,2 Lei Qian Tai,1,2 Jacques-Olivier Bay,2,3 and Richard A. Gatti1,2*1Jules Stein Eye Institute, UCLA School of Medicine, Los Angeles, California2Department of Pathology, UCLA School of Medicine, Los Angeles, California3Laboratoire d’Oncologie Moleculaire, INSERM 9402 et 2145, Centre Jean Perrin, Clermont-Ferrand, France

We have found a complex repeat sequence(NS22) that is highly polymorphic and lo-cated within intron 45 of the ataxia-telan-giectasia gene (ATM). Sequencing this re-gion from various individuals demonstratedtwo different polymorphic repeating unitsadjacent to one another. The fact that thesequence is located within the ATM geneprovides a unique opportunity to follow seg-regation of affected and unaffected haplo-types for prenatal diagnosis of ataxia-telangiectasia. The high degree of polymor-phism observed with this marker will alsoaid in evaluating loss of heterozygosity(LOH) across this region of the genome andmay prove valuable in assessing the role ofthe ATM gene in susceptibility to cancer.Am. J. Med. Genet. 82:287–289, 1999.© 1999 Wiley-Liss, Inc.

KEY WORDS: microsatellite; STR; complexrepeat; ATM; A-T gene

INTRODUCTION

The important characteristics of a genetic markerare informativeness and ease in testing so that it canbe used to follow segregation, linkage disequilibrium,loss of heterozygosity (LOH), or uni-parental disomy.The marker, NS22, satisfies these criteria. Because ofits location within the ATM gene, it is especially usefulfor prenatal diagnosis of ataxia-telangiectasia (AT) andfor following the extent of LOH within the 11q22-23region. Because of its complex repeats it is only of lim-ited use in haplotyping a general population.

AT is an autosomal recessive neurological disorder of

childhood. The phenotype includes progressive cerebel-lar ataxia, telangiectasia, recurrent infections, chromo-somal instability, cancer susceptibility, and radiationsensitivity. An international consortium mapped theATM gene by linkage analysis to within a 500-kb in-terval on chromosome 11q22.3-23.1 [Gatti et al., 1988;Lange et al., 1995]. Savitsky et al. [1995] isolated thegene from this region.

MATERIALS AND METHODS

Primer sequences were ATM7F, AGGAATATG-CAGTGGGACCAT; NS22F, TCTTCTATGAATATAA-CAGGAGTTGTT; and NS22R, GCTTGCAACATCTAC-TATATATTTTTT. The sizes of the polymerase chainreaction (PCR) products were 264-292 bp for ATM7F–NS22R and 156–184 bp for NS22F–NS22R. The PCRreaction mixture contained dNTP (1.25 mM) 1.0 ml, 10× PCR buffer I (Perkin Elmer, Norwalk, CT) 1.0 ml,labeled primer (10ng/ml) 0.5 ml, unlabeled complemen-tary primer (100ng/ml) 0.05 ml, DNA template (500 ng/ml) 1.0 ml, Taq DNA polymerase (5u/ml) 0.1 ml in a totalvolume of 10 ml. One microliter of primer (100ng/ml)was labeled by adding 5× kinase buffer 1.0 ml, T4 poly-nucleotide kinase (10u/ml) 1.0 ml, g33PdATP (3000Ci/mmol) 3.0 ml, and water to a total volume of 10 ml. Themixture was incubated at 37°C for 10 min and then theenzyme was denatured by heating at 94°C for 2 min.Cycling was for 18 cycles: 94° for 30 sec, 57° for 30 sec,72° for 30 sec, and an extension of 72° for 10 min. ThePCR products were electrophoresed on a 6% denatur-ing polyacrylamide gel with 7 M urea in 1 × TBE. PCRproducts were sequenced using a Thermo Sequenaseradiolabeled terminator cycle sequencing kit (Amer-sham, Buckinghamshire, UK).

RESULTS AND DISCUSSION

We found repeat sequences located adjacent to oneanother between 105159–105423 bp of the ATM gene[Platzer et al., 1997] (Fig. 1). We synthesized threeprimers flanking this repeat sequence. We establishedMendelian segregation in CEPH families 1333, 1334(Fig. 2), and 1340. The sequenced alleles of CEPH[Dausset et al., 1990] members 1333-12 and 1340-10are shown in Figure 3.

Contract grant sponsor: AT Medical Research Foundation;Contract grant sponsor: U.S.Public Health Service; Contractgrant number: NS35322.

*Correspondence to: Dr. Richard A. Gatti, Dept. of Pathology,UCLA School of Medicine, 10833 Le Conte Ave., Los Angeles, CA90095. E-mail: RGatti@pathology.medsch.ucla.edu

Received 9 February 1998; Accepted 12 October 1998

American Journal of Medical Genetics 82:287–289 (1999)

© 1999 Wiley-Liss, Inc.

We observed 72% heterozygosity in the general popu-lation with NS22 (48 of 67 unrelated persons). We se-quenced 13 individuals across the region of the repeatsin order to determine which of the repeating units werepolymorphic and what their frequencies were. Wefound that the first two repeating units,(TA) and (CA),were polymorphic. Individual 1333-12 has (TA)15 re-peats and (CA)11 repeats, while individual 1334-10 had(TA)23 repeats and (CA)12 repeats (Fig. 3). This highlyinformative marker located within the ATM gene canbe of great advantage for diagnostic purpose. For pre-natal testing, the two affected haplotypes are estab-

lished on prior affected members; the segregation of theaffected alleles can then be followed, since the ATMgene is too large for detecting mutations by direct se-quencing (66 exons). Over 250 mutations have beenobserved all across the gene and almost all patientswho are not consanguineous carry two different ATMmutations [Telatar et al., 1996; Concannon and Gatti,1997; Telatar et al., 1998; website http://www.vmmc.org/vmrc/atm.htm]. Thus, finding mutations in everyaffected individual is difficult and time consuming;

Fig. 2. Segregation of NS22 alleles within CEPH family 1334. Gel pic-ture under the pedigree shows the alleles for each individual. Numbers ontop of the lanes designate individual numbers. Lane 3 has no DNA.

Fig. 1. Sequence of the ATM gene with repeats. The polymorphic re-peating units are in capital letters, primer names in bold, and primersequences underlined.

Fig. 3. DNA sequence for the NS22 marker. Individual CEPH 1333-12shows from top the complementary polymorphic repeat (AT)15 followed by(GT)11 and individual CEPH 1340-10 with (AT)23 followed by (GT)12. Thepolymorphic repeating units are between the two arrows.

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knowledge of the affected haplotypes can be usefuleven before the actual mutations have been defined.

What makes NS22 especially informative is that ithas two different adjacent repeating units that areboth polymorphic, thus allowing a highly informativemini-haplotype to be created. On the other hand, be-cause the marker has two polymorphic repeats withinone PCR product, it is important to note that the size ofthe allele will reflect the size of two independent repeatalleles. Thus, unrelated individuals with alleles of thesame size cannot be equated automatically and allelicfrequencies cannot be determined without sequencingeach person. On the other hand, alleles of differentsizes will still allow different haplotypes to be distin-guished.

The location of this marker should also be useful forloss of heterozygosity (LOH) studies across the 11q22-23 region, a region where LOH has been associatedwith melanomas, breast and prostate cancers, and leu-kemia [Foulkes et al., 1993; Gustafson et al., 1994;Bethwaite et al., 1995; Hampton et al., 1994; Negrini etal., 1995; Rasio et al., 1995; Herbst et al., 1995; Laakeet al., 1997; Rio et al., 1998]. Because several othergenes exist in close proximity to the ATM gene, usingmarkers located outside the ATM gene does not distin-guish whether LOH includes or excludes the ATMgene. Using NS22 now makes this possible. Further,another marker, D11S2179, is also located within theATM gene. Although not very informative by itself, itcan now be used in conjunction with NS22 to yield verydistinct and informative haplotypes.

ACKNOWLEDGMENTS

This work was supported by the AT Medical Re-search Foundation, U.S. Department of Energy grant87ER60548, and USPHS grant no. NS35322.

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