Seven DNA variants that polymorphic genetic marker D16S752 reveals in Croatian
population are reported in this paper. The marker is a GATA tetranucleotide repeat
linked to human E-cadherin gene (CDH1). Prior studies involving this marker revealed
only four DNA allele variants. The reported DNA variants contribute to the collection of
hypervariable DNA polymorphisms data useful in the field of anthropological and population
genetic and forensic medicine

J. Pavelić

K. Gall-Trošelj

K. Pavelić

N. Pećina-Šlaus

S. Kapitanović

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Novel Alleles of the D16S752 Polymorphic Genetic Marker Linked to E-Cadherin Gene – A Potential Population Marker

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Coll. Antropol. 26 (2002) 1: 85–88UDC 572:575.174.015.3Original scientific paperNovel Alleles of the D16S752Polymorphic Genetic Marker Linkedto E-Cadherin Gene – A PotentialPopulation MarkerN. Pe}ina-[laus1, K. Gall-Tro{elj2, S. Kapitanovi}2, J. Paveli}2and K. Paveli}21 Department of Biology, School of Medicine, University of Zagreb, Zagreb, Croatia2 Division of Molecular Medicine, »Ru|er Bo{kovi}« Institute, Zagreb, CroatiaA B S T R A C TSeven DNA variants that polymorphic genetic marker D16S752 reveals in Croatianpopulation are reported in this paper. The marker is a GATA tetranucleotide repeatlinked to human E-cadherin gene (CDH1). Prior studies involving this marker revealedonly four DNA allele variants. The reported DNA variants contribute to the collection ofhypervariable DNA polymorphisms data useful in the field of anthropological and pop-ulation genetic and forensic medicine.IntroductionTetranucleotide repeat D16S752 (GATA-51G03) is a polymorphic marker linked totumor suppressor gene E-cadherin (CDH1)(16q22.1)1 which encodes a calcium-de-pendent cell adhesion molecule involvedin epithelial development and mainten-ance2.Polymorphic markers have proved tobe very useful in tumor allelotyping anddetection of loss of heterozygosity (LOH)of putative tumor suppressor genes3. Inthis respect D16S752 marker has beenused for LOH detection of CDH1 gene incarcinomas. Nevertheless, polymorphismshave found extensive application in anthro-pological and population genetic studiesproviding data for the genetic structure ofdifferent populations4–5. For this purposeit is very important to know all allelicvariants this marker could reveal in nor-mal tissue of certain population.Here we report on novel allelic vari-ants of D16S752 found in constitutiveDNA from unrelated individuals.85Received for publication December 15, 2001Materials and MethodsDNA was isolated from blood samplesof unrelated individuals (54) from Croa-tia suffering from renal or colon carci-noma or phaeochromocytoma. The poly-morphic region was PCR amplified (totalvolume – 25 l, each primer (5'-AAT-TGACGGTATATCTATCTGTCTG-3'; and5'-GATTGGAGGAGGGTGATTCT-3') 5 pmol,200 ng DNA, 2.5 l 10X buffer II, 1.5 mMMgCl2, 2.5 mM of each dNTP, 0.25 U Taqpolymerase; Eppendorf, Germany). PCRconditions: initial denaturation, 3 min/96°C; denaturation, 30 s/96 °C; annealing,35 s/55 °C; extension, 30+1 s/72 °C; finalextension, 72 °C/10 min; 35 cycles.Each polymorphism was analyzedboth on 15% polyacrylamide gels run in1X TBE (Tris/borate/EDTA, stained withsilver) and on Spreadex EL 300 gels (El-chrom scientific, Cham, Switzerland).The electrophoresis conditions were:0,75XTAE (Tris/acetate/EDTA), 120min/120V, 55 °C. Spreadex gels were Syber-Gold stained (Molecular Probes, Leiden,Netherlands).All the procedures employed were ap-proved by the appropriate review com-mittee.ResultsIn a random sample of constitutiveDNA from unrelated individuals we foundseven different allelic variants of poly-morphic marker D16S752; three of themyet not described in the literature.From 54 analyzed samples 48 wereheterozygous which showed that D16S-752 polymorphism has mean observedand expected heterozygosity values of 89%and 92%, respectively.Individual allele frequencies are shownin Table 1. The alleles were each assignedby numbers 1 to 7. The allele number 2(106 bp) appeared most frequent, follo-wed by alleles 3 (110 bp) and 4 (114 bp), 5(118 bp), 1 (102 bp), 6 (122 bp) and 7 (126bp).Figure 1 demonstrates all seven allelicvariants. By comparison to molecularmarker we calculated that each allele dif-fers by four nucleotides from the preced-ing one.DiscussionIn constitutive DNA samples fromCroatia we noticed additional number ofalleles in regard to D16S752 genetic mar-ker. Sex and age of the subjects showedno correlation to the allele distribution.86N. Pe}ina-[laus et al.: Novel Alleles of D16S752 Marker, Coll. Antropol. 26 (2002) 1: 85–88TABLE 1ALLELE FREQUENCIES AND THEIR COMBINATIONS IN HETEROZYGOUS INDIVIDUALSAllele Total allele frequency Allelecombination*Number ofindividualsAllelecombination*Number ofindividualsHeterozygous Homozygous1 10 0 1, 2 5 3, 4 62 18 4 1, 3 2 3, 5 33 17 4 1, 5 3 3, 6 24 19 2 2, 2 2 4, 4 15 16 2 2, 3 4 4, 5 26 10 0 2, 4 3 4, 6 37 6 0 2, 5 4 4, 7 52, 6 1 5, 5 12, 7 1 5, 6 43, 3 2*None of the samples tested had combinations 1/1, 1/4, 1/6, 1/7, 3/7,5/7, 6/6, 6/7, 7/7.According to the GenomeDataBase(http://www.gdb.org/) only four D16S752allele variants were reported so far.Moreover, they were scored on northernEuropean populations and very few peo-ple were tested (unpublished communica-tion).Additional number of alleles, in re-gard to D16S752, scored in samples ob-tained from Croats and as a consequence,high frequency of their heterozygoticcombination may be helpful for furthergenetic studies on the role of CDH1 in tu-mor development and progression.Measuring rates of genetic divergenceamong populations as well as establish-ing genetic markers characteristic forcertain population is one of the promisinguses of reported DNA variants in the fieldof anthropology and population genetic6–7as well as tumor allelotyping. Moreover,specific markers are useful for resolvinggenetic patterns of variability at regionallevels8. The data presented in this paperbroaden the genetic spectrum and con-tribute to our knowledge of the geneticnature of the Croatian population.AcknowledgmentThis work was supported by grants00981104 and 0108509 from Ministry of Sci-ence and Technology, Republic of Croatia.R E F E R E N C E S1. BERX, G., K. STAESS, J. VAN HENGEL, F.MOLEMANS, M. J. BUSSEMAKERS, A. VAN BOK-HOVEN, F. VAN ROY, Genomics, 26 (1995) 281. — 2.GUILFORD, P., Mol. Med. Today, 5 (1999) 172. — 3.KORETH, J., J. J. O'LEARY, J. O. D. McGEE, J.Pathol., 178 (1996) 239. — 4. MARTINOVI], I., L.BARA], I. FURAC, B. JANI]IJEVI], M. KUBAT, M.PERI^I], B. VIDOVI], P. RUDAN, Hum Biol., 71(1999) 341. — 5. KLARI], I. M., L. BARA], D. BU-KOVI], I. FURAC, G. GEBER, B. JANI]IJEVI], M.KUBAT, M. PERI^I], B. V. PUPI], P. RUDAN, AnnHum Biol., 28 (2001) 281. — 6. PARKER, P. G., A. A.SNOW, M. D. SCHUG, G. C. BOOTON, P. A.FUERST, Ecology, 79 (1998) 361. — 7. KRUL, E. J. T.,A. F. KERSEMAEKERS, Y.A. ZOMERDIJK-NOOY-EN, C. J. CORNELISSE, L. A. W. PETERS, G. J.FLEUREN, Cancer, 86 (1999) 997. — 8. TOLK, H. V.,L. BARA], M. PERI^I], I. M. KLARI], B. JANI]I-JEVI], H. CAMPBELL, I. RUDAN, T. KIVISILD, R.VILLEMS, P. RUDAN, Eur. J. Hum. Genet., 9 (2001)717.87N. Pe}ina-[laus et al.: Novel Alleles of D16S752 Marker, Coll. Antropol. 26 (2002) 1: 85–88Fig. 1. Seven different D16S752 allele variants. Spreadex gel, stained with SyberGold (MolecularProbes). Lane 1– molecular marker M3 (Elchrom Scientific); lanes 2, 6 – alleles 2/5; lanes 3, 5 – al-leles 4/7; lane 4 – alleles 5/6; lane 7 – alleles 3/5; lane 8 – alleles 3/4; lanes 9, 10 – homozygoussamples, alleles 5/5; lanes 11, 12 – alleles 4/5; lane 13 – alleles 1/2.N. Pe}ina-[lausDepartment of Biology, School of Medicine, University of Zagreb, [alata 3,10000 Zagreb, CroatiaNOVI ALELI POLIMORFNOG GENETI^KOG MARKERA D16S752:POTENCIJALNI POPULACIJSKI MARKERS A @ E T A KUpotrebom polimorfnog geneti~kog markera D16S752 otkriveno je sedam DNA va-rianti u populaciji Hrvatske. Marker je tetranukleotidno GATA ponavljanje vezano uzgen E-cadherin (CDH1) ~ovjeka. Prija{nja istra`ivanja ovim markerom pokazala susamo ~etiri razli~ita alela. Prikazani polimorfizam vrijedan je podatak za prikupljanjeDNA polimorfizama neophodnih u antropolo{koj i populacijskoj genetici, te sudskojmedicini.88N. Pe}ina-[laus et al.: Novel Alleles of D16S752 Marker, Coll. Antropol. 26 (2002) 1: 85–88

Novel Alleles of the D16S752 Polymorphic Genetic Marker Linked to E-Cadherin Gene – A Potential Population Marker

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