POLYMORPHISM OF THE STR LOCI: TH01, TPOX AND CSF1PO IN THE POPULATION OF UPPER SILESIA; THEIR USEFULNESS IN DISPUTED PATERNITY TESTING

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1 POLYMORPHISM OF THE STR LOCI: TH01, TPOX AND CSF1PO IN THE POPULATION OF UPPER SILESIA; THEIR USEFULNESS IN DISPUTED PATERNITY TESTING Ewa RACZEK, Kornelia DRO DZIOK, Jadwiga KABIESZ Chair and Deartment of Forensic Medicine, Silesian Medical Academy, Katowice ABSTRACT: Poulation genetics studies were carried out on samles of unrelated individuals from the Uer Silesian oulation using STR systems: TH01 (n = 240), TPOX and CSF1PO (n = 162). No deviations from the H-W equilibrium were observed. No rare alleles and no aberrations from Mendelian laws were found. The allele frequency at two (TPOX, CSF1PO) of the three STR loci in the Uer Silesian oulation was similar to those of other Polish and Euroean oulations. Allele frequencies of Polish oulations which had been comared with each other in the TH01 system were not homogeneous. The following statistical arameters of forensic imortance were calculated: observed heterozygosity (Htobs.), exected heterozygosity (Htex) discrimination ower (PD), mean exclusion chance (MEC), mean exclusion robability (MEP) and olymorhic information content (PIC). KEY WORDS: Polymorhism; TH01; TP0X; CSF1PO, Uer Silesia. Z Zagadnieñ Nauk S¹dowych, z. XLV, 2001, Received 22 January 2001; acceted 11 Aril 2001 INTRODUCTION Systems: TH01 (1115.5), TPOX (2113-2ter) and CSF1PO (5q33.5-q34) are highly olymorhic STR tye DNA loci. In all human races, these systems have a well-defined olymorhism level and are useful for DNA analysis carried out in criminalistic and disuted aternity investigations [1, 2, 3, 4, 5, 6, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 21, 22]. The aim of this work was to determine the genotye frequency of the TH01, TPOX, and CSF1PO loci in the oulation of Uer Silesia and demonstrate its homogeneity with oulations from other regions of Poland.

2 82 E. Raczek, K. DroŸdziok, J. Kabiesz MATERIALS AND METHODS Polymorhism analysis of TH01, TPOX and CSF1PO loci was carried out on a samle of unrelated adults living in the Uer Silesia region. In the resent study the THO1 locus was examined in 240 individuals, and the TPOX and CSF1PO loci in 162 individuals. DNA was extracted using a modified version of the Kunkel method [7] instead of henol, a chloroform: isoamyl alcohol (24:1) mixture was used. The concentration and urity of the extracted DNA was assessed by the sectrohotometric method using Hitachi aaratus and the DNA/rotein card 1E rogramme. Amlification was carried out using a Perkin Elmer 480 thermocycler (Perkin Elmer). PCR roducts were searated in a 6% denaturing vertical olyacrylamide gel (Gene Page Plus-Amresco) using SA-32 aaratus (Life Technologies) and silver stained. The amlification, elctrohoresis and silver staining was erformed according to the rotocol sulied with the amlification kit, including single locus TH01 amlification system and trilex CSF1PO, TPOX and TH01 amlification kit, made by Promega cororation. Statistical calculations were erformed using TFPGA (exected and observed heterozygosity and exact tests) and Carmody s (homogeneity test) software, kindly submitted by Mrs P. Wolañska-Nowak, MSc (Institute of Forensic Research in Cracow) and Dr P. Kozio³ (Faculty of Legal Medicine in Lublin). RESULTS AND DISCUSSION The olymorhism level of analysed systems is resented in Table I. The resence of 6 out of 8 alleles described in the literature [5] and 15 henotyes were observed at the TH01 locus in the Uer Silesian oulation. 5 out of 8 [4] and 7 out of 10 [8, 19] alleles described in the literature were found at the TPOX and CSF1PO loci resectively. For the TPOX and CSF1PO loci, 11 and 16 henotyes resectively were revealed. In the Uer Silesian oulation, the most frequently observed allele of the TH01 locus is 9.3 (f = ), and the least frequently allele 10 (f = ). The most frequent henotyes, and 9-9.3, occurred with frequency f = , and thus can be stated to be widesread. Allele 8 (f = ) and 11 (f = ), and henotyes 8-8 (f = ) and 8-11 (0.2469) dominate at locus TPOX in the studied oulation. Although the CSF1PO locus is considered much more diverse (10 described alleles), the most frequent alleles are 12 (f = ) and 10 (f = ), whilst the most

3 Polymorhism of the STR loci: TH01, TPOX commonly observed henotyes are (f = ) and (0.1605). Inter-alleles were not observed at any of the analysed loci. TABLE I. FREQUENCIES OF LOCI: TH01, TPOX AND CSF1PO IN THE POPULATION OF UPPER SILESIA Allele TH01 n = 240 TPOX n = 162 CSF1PO n = Test df Exact test (Markov Chain) Exact test (Monte Carlo) 15 = = = ± = ± = = = ± = ± = = = ± = ± Similarly to other reorted Polish, Euroean and world oulations, the TH01, TPOX and CSF1PO loci in the Uer Silesian region show no deviations from Hardy-Weinberg equilibrium, as shown by the test and the exact test, calculated using the Monte Carlo method and Markov chain (TFPGA software, Table I) [1, 2, 3, 4, 5, 6, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 21, 22]. A comarison of the allele frequencies of analysed loci in the Uer Silesian oulation with other regions of Poland is resented in Table II. The comarison in Table II indicates that for a confidence level = 0.05, only the oulations of north-east Poland [13, 14], Pomorze-Kujawy [8] and Lower Silesia [3] have a similar distribution of allele frequencies to Uer Silesia. The remaining reorted Polish oulations show differences in allele frequencies at the TH01 locus. A similar lack of concordance to the Uer Silesian oulation in terms of allele frequencies of the TH01 system ( < 0.05 in Carmody test) was found for German [6], Slovenian [22] and Sanish [4] oulation samles. Only the Italian [5] oulation revealed a similar distribution of allele frequencies at TH01 ( = 0.015) to the Uer Silesian region. Additionally, a lack of concordance in alleles distribution was observed between the oulation of Uer Silesia and that of, for examle, Jaan or Zimbabwe ( = 0.000) [2, 11].

4 84 E. Raczek, K. DroŸdziok, J. Kabiesz TABLE II. COMPARISON OF THE ALLELE FREQUENCIES OF LOCI: TH01, TPOX AND CSF1PO IN THE POPULATION OF UPPER SILESIA TO OTHER POLISH POP- ULATION SAMPLES TH01 TPOX CSF1PO Uer Silesia (own versus: North Poland [12] No North Poland [17, 18] No Yes Yes North-east Poland [13, 14] Comared oulations Pomerania- Kujawy [8] Yes Yes Yes Yes Yes Yes Lower Silesia [3] Yes Yes Yes South Poland [19] No Yes Yes South Poland [21] No Yes Yes South-east Poland [9] No Yes No Yes 0.05; No < Some Polish regional oulations show a lack of homogeneity at the TH01 locus, when comared each one with each one and all-together (Table III). TABLE III. HOMOGENEITY OF POLISH POPULATIONS (CARMODY S TEST) Comared oulations TH01 TPOX CSF1PO North Poland [12] versus: North Poland [17, 18] Yes North-east Poland [13, 14] Yes Pomerania-Kujawy [8] Yes Lower Silesia [3] Yes Uer Silesia (own No South Poland [19] Yes South Poland [21] Yes South-east Poland [9] No North-east Poland [13, 14] North Poland [17, 18] versus: Yes Yes Yes Pomerania-Kujawy [8] Yes Yes Yes Lower Silesia [3] Yes Yes Yes Uer Silesia (own No Yes Yes South Poland [19] Yes Yes Yes South Poland [21] Yes Yes Yes South-east Poland [9] No Yes Yes

5 Polymorhism of the STR loci: TH01, TPOX TABLE III. Continuation Comared oulations TH01 TPOX CSF1PO North-east Poland [13, 14] versus: Pomerania-Kujawy [8] Yes Yes Yes Lower Silesia [3] Yes Yes Yes Uer Silesia (own Yes Yes Yes South Poland [19] Yes Yes Yes South Poland [21] Yes Yes Yes South-east Poland [9] No Yes Yes Pomerania-Kujawy [8] versus: Lower Silesia [3] Yes Yes Yes Uer Silesia (own Yes Yes Yes South Poland [19] Yes Yes Yes South Poland [21] Yes Yes Yes South-east Poland [9] No No Yes Uer Silesia (own Lower Silesia [3] versus: Yes Yes Yes South Poland [19] Yes No Yes South Poland [21] Yes No Yes South-east Poland [9] No No No Uer Silesia (own South Poland [19] versus: No Yes Yes South Poland [21] Yes Yes Yes South-east Poland [9] Yes No Yes Uer Silesia (own South Poland [21] versus: No Yes Yes South-east Poland [9] Yes Yes Yes Uer Silesia (own South-east Poland [9] versus: No Yes No Poland (simultaneous comarison of oulation samles from different regions) No Yes Yes Yes 0.05; No < Analysing the results of the TH01 locus homogeneity test alied here, one can conclude that significant differences are caused by: different frequencies of occurrence of the three closely migrated alleles, 9, 9.3 and 10, in regional oulations [9, 19, 21] and different frequency of the widesread alleles 6 and 9.3 [19, 21]. Difficulties in correct labelling of alleles 9.3 and 10 may be the cause of lack of homogeneity between oulations of southern Po-

6 86 E. Raczek, K. DroŸdziok, J. Kabiesz land (table III). The APLP (the amlified roduct length olymorhism) method described in the literature [20], which uses a set of three rimers for amlification of locus TH01 and leads to fast, unambiguous differentiating of alleles 9, 9.3 and 10, may hel to avoid this sort of divergence in the future. Allele 10 occurs in south-east Poland with a frequency of [9], whilst in Uer Silesia with a much lower frequency of , which is closer to the remaining Polish and Euroean oulations [4, 5, 6, 8, 13, 14]. Frequencies obtained for alleles 6 and 9.3 in southern Poland [19, 21] are similar. In other regions in Poland, however, allele 9.3 is most frequent [3, 8, 9, 13], which may also contribute to lack of homogeneity of allelic frequencies at locus TH01 calculated for the whole Polish oulation (in Carmody s homogeneity test all oulations of Poland were comared at the same time Table III). Disroortions in frequency of occurrence of alleles 6 and 9.3 in different regions of Poland can not be exlained by their incorrect henotying; an interretation error in this case is unlikely. However, the observed statistically significant divergences can not be exlained only by regional differences (suboulations), or by the small number of ersons studied. One should also take into consideration, for examle, incorrect allele tying using commercially available Promega allele ladders (the original ladder does not include allele 9.3) and the imerfect system of amlification roducts detection. There is a significant concordance between the two oulation samles from northern Poland [12, 17] and southern Poland [19, 21] analysed in indeendent laboratories. Perfect concordance can also be observed at loci TPOX and CSF1PO for oulation samles from the Cracow area [19, 21]; (Table III). Differences between regions, however, were not found for allele frequency at locus TPOX, which is much easier to henotye correctly and less variable. At this locus, the oulation samle from Uer Silesia is homogeneous with other Polish oulations (Table II), and with Euroean oulation samles ( > 0.05) [4, 5, 6, 22], but significantly divergent from Asian and Negroid oulations ( = 0.000) [2, 11]. Comarative analysis of all Polish oulations, however, revealed differences in frequencies at locus TPOX between some Polish oulation samles. Data obtained for the south-east region of Poland [9] are not consistent with data for the Pomerania-Kujawy region [8], southern Poland [19] and Lower Silesia. Allele distributions at locus TPOX for Lower Silesia also differ from those of southern Poland [19, 21]. These differences, however, do not cause a lack of homogeneity of allele frequencies at locus TPOX for the whole Polish oulation (Table III). In turn allele frequencies for locus CSF1PO in oulations of Uer Silesia and other researched regions of Poland are homogeneous relative to each other, with one excetion south-eastern Poland [9] (Table II). Allele frequencies obtained for locus CSF1PO are also homogeneous in most of the

7 Polymorhism of the STR loci: TH01, TPOX Polish oulation samles (Table III). Comarative analysis of allele frequencies obtained for this locus in studied oulation samles revealed that South-East Poland [9] is significantly divergent from Lower and Uer Silesia [3]. These differences do not influence the homogeneity of the CSF1PO allele frequencies in all studied oulation samles from Poland (Table III). Statistical data imortant for aternity testing calculated for the three analysed STR loci are resented in Table IV. TABLE IV. STATISTICAL PARAMETERS DESCRIBING THREE ANALYSED LOCI: TH01, TPOX AND CSF1PO IN THE POPULATION SAMPLE OF UPPER SILESIA STR system PD MEC MEP PIC Ht obs Ht ex TH TPOX CSF1PO TH01 TPOX CSF1PO PD ower of discrimination; MEC mean exclusion chance (usefulness of the system); MEP mean aternity exclusion robability; PIC olymorhism information content; Htobs observed heterozygosity; Htex exected heterozygosity. These data are not divergent from results obtained for other regions in Poland [3, 8, 9, 12, 13, 14, 17, 18, 19, 21] and Euroe [4, 5, 6, 22]. The total ower of discrimination (PD) and mean exclusion chance (MEC), also known as the usefulness of the system (PE), for studied loci equal and resectively. Such values confirm the great usefulness of the systems in disuted aternity cases. Among 111 such cases for which analysis of the trilex CTT (73) or the monolex TH01 (38) was erformed, no mutations or deviations from rules of heredity were noted. However, 10 exclusions of wrongly named men as fathers were noted, among whom in 8 cases exclusion was on the basis of results obtained for TH01, TPOX or CSF1PO STR systems. Most frequently exclusion of the non-fathers was done according to data obtained for locus TH01 (8 cases), less frequently for locus CSF1PO (6 cases) and TPOX (3 cases). These facts, and also the characterisation of usefulness of articular loci in disuted aternity testing resented in Table IV, jointly confirm that trilex CTT is the most useful, and that the most useful individual system is locus TH01.

8 88 E. Raczek, K. DroŸdziok, J. Kabiesz References: 1. A s m u n d o A., C r i n ó C., Poulation study of the short tandem reeat olymorhisms HumTH01, HumvWA31, HumFESFPS and HumF13A01 in Sicily (Southern Italy), International Journal of Legal Medicine 1998, vol. 111, B u d o w l e B., N h a r i L. T., M o r e t t i T. R. [et al.], Zimbabwe black oulation data on the six short tandem reeat loci CSF1PO, TPOX, TH01, D3S1358, VWA and FGA, Forensic Science International 1997, vol. 90, D o b o s z T., Czêstoœci wybranych genów warunkuj¹cych olimorfizm genetyczny w obrêbie oulacji dolnoœl¹skiej, Zeszyty Naukowe Akademii Medycznej we Wroc³awiu 1999, t. 74, s G a r c i a O., M a r t i n P., B u d o w l e B. [et al.], Basque Country autochthonous oulation data on 7 short tandem reeat loci, International Journal of Legal Medicine 1998, vol. 111, G a r o f a n o L., P i z z a m i g l i o M., V e c c h i o C. [et al.], Italian oulation data on thirteen short tandem reeat loci: HUMTH01, D21S11, D18S51, HUMVWFA31, HUMFIBRA, D8S1179, HUMTPOX, HUMCSF1PO, D16S539, D7S820, D13S317, D5S818, D3S1358, Forensic Science International 1998, vol. 97, H a n t s c h e l M., H a u s m a n n R., L e d e r e r T. [et al.], Poulation genetics of nine short tandem reeat (STR) loci DNA tying using the AmFISTR Profiler PCR amlification kit, International Journal of Legal Medicine 1999, vol. 112, K u n k e l L. M., S m i t h K. D., B a y e r S. H. [et al.], Analysis of human Y-chromosome secific reiterated DNA in chromosome variants, Proceedings of National Academy of Sciences USA 1977, vol. 74, M i œ c i c k a - Œ l i w k a D., C z a r n y J., G r z y b o w s k i T. [et al.], Poulation genetics of 14 STRs: vwa, TH01, TPOX, CSF1PO, D5S818, D13S317, D7S820, D16S539, F13A01, FESFPS, F13B, LPL, D3S1358, and FGA in the Pomerania-Kujawy region of Poland, [in:] Olaisen B., Brinkmann B., Lincoln P. J. [eds.], Progress in forensic genetics, Excerta Medica, Amsterdam 1998, M o n i e s D., C i e s i e l k a M., K o z i o ³ P. [et al.], Czêstoœci alleli szeœciu uk³adów STR (CSF1PO, TPOX, TH01, F13A01, FESFPS, VWA) w oulacji Polski o³udniowo-wschodniej, Postêy Medycyny S¹dowej i Kryminologii 1997, t. 3, s N a g a i A., Y a m a d a S., W a t a n a b e Y. [et al.], Analysis of the STR loci HUMF13A01, HUMFXIIIB, HUMLIPOL, HUMTH01, HUMTPOX and HUMVWFA31 in a Jaanese oulation, International Journal of Legal Medicine, 1996, 109, N a t a M., K i m u r a T., H a s h i y a d a M. [et al.], Allele frequencies of eight STRs in Jaanese and Chinese, International Journal of Legal Medicine 1999, vol. 112, P a w ³ o w s k i R., M a c i e j e w s k a A., P a s z k o w s k a R. [et al.], Frequencies for five short tandem reeat (STR) systems in a oulation from North Poland, International Journal of Legal Medicine 1997, vol. 110,

9 Polymorhism of the STR loci: TH01, TPOX P e i ñ s k i W., S k a w r o ñ s k a M., K o c E. [et al.], Polimorfizm uk³adu TH01 w oulacji Polski ó³nocno-wschodniej, Postêy Medycyny S¹dowej i Kryminologii 1999, t. 5, s P e i ñ s k i W., J a n i c a J., N i e m c u n o w i c z - J a n i c a A. [et al.], Genetyka oulacyjna 9 uk³adów tyu STR w regionie Polski ó³nocno-wschodniej, Streszczenia z I Symozjum Hemogenetyki S¹dowej, Pieczyska/Bydgoszcz, maja 2000 r. 15. P u C. E., W u F. C., C h e n g C. L. [et al.], DNA short tandem reeat rofiling of Chinese oulation in Taiwan determined by using an automated sequencer, Forensic Science International 1998, vol. 97, S u e b l i n v o n g T., K o n g s r i s o o k U., Poulation data of 8 short tandem reeat loci in the Thai oulation, Forensic Science International 1999, vol. 103, S z c z e r k o w s k a Z., W y s o c k a J., Polimorfizm dwóch loci STR: HUMTPOX i HUMTH01. Zastosowanie w dochodzeniu ojcostwa, Annales Academiae Medicae Gedanensis 1999, vol. 29, s S z c z e r k o w s k a Z., W y s o c k a J., K a i ñ s k a E., Polimorfizm dwóch loci HUMF13A01 i CSF1PO. Badania oulacyjne, zastosowanie w medycynie s¹dowej, Annales Academiae Medicae Gedanensis 2000 [w druku]. 19. T u r o w s k a B., S a n a k M., Badanie czêstoœci alleli niektórych systemów tyu STR w oulacji Polski o³udniowej, Archiwum Medycyny S¹dowej i Kryminologii 1998, t. XLVIII, s W a t a n a b e G., U m e t s u K., S u z u k i T., Determination of the HUMTH01 alleles by the APLP method, International Journal of Legal Medicine 1999, vol. 112, W o l a ñ s k a - N o w a k P., Alication of suboulation theory to evaluation of DNA evidence, Forensic Science International 2000, vol. 113, Z u a n i è I., B a l a i è J., K o m e l R., Analysis of nine short tandem reeat (STR) loci in the Slovenian oulation, International Journal of Legal Medicine 1998, vol. 111,

10 POLIMORFIZM LOCI TYPU STR: TH01, TPOX I CSF1PO W POPULACJI GÓRNEGO ŒL SKA; ICH PRZYDATNOŒÆ DO BADAÑ SPORNEGO OJCOSTWA Ewa RACZEK, Kornelia DRO DZIOK, Jadwiga KABIESZ WPROWADZENIE Uk³ady: TH01 (1115.5), TPOX (2113-2ter) i CSF1PO (5q33.5-q34) to wysoce zró nicowane loci DNA tyu STR. Ich olimorfizm i rzydatnoœæ do badañ sornego ojcostwa oraz do badañ kryminalistycznych wykazano dla wszystkich ras [1, 2, 3, 4, 5, 6, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 21, 22]. Niniejsz¹ racê odjêto w celu rzedstawienia czêstoœci genowych w tych trzech loci w oulacji Górnego Œl¹ska i wykazania jej homogennoœci z innymi regionami Polski. MATERIA I METODYKA Polimorfizm loci: TH01, TPOX i CSF1PO oznaczano wœród niesokrewnionych osób doros³ych z Górnego Œl¹ska (dla lokus TH01 badaniami objêto 240 osobników; dla loci TPOX i CSF1PO 162). Izolacjê DNA rowadzono zmodyfikowan¹ metod¹ Kunkela i in. [7]. Modyfikacja olega³a na zast¹ieniu fenolu mieszanin¹ chloroform : alkohol izoamylowy w stosunku objêtoœciowym 24:1. Stê enie i czystoœæ DNA oznaczano sektrofotometrycznie (sektrofotometr Hitachi, rogram DNA/rotein card 1E). Amlifikacjê (amlifikator ty 480 firmy Perkin Elmer), rozdzia³ elektroforetyczny (aarat do elektroforezy wertykalnej ty SA-32 firmy Life Technologies, 6% denaturuj¹cy el Gene Page Plus firmy Amresco) i barwienie azotanem srebra wykonywano zgodnie z zaleceniami firmy Promega, roducenta zestawu do amlifikacji alleli ojedynczego lokus TH01 oraz trzech loci CSF1PO, TPOX i TH01. Obliczenia statystyczne rzerowadzono w oarciu o rogramy komuterowe: TFPGA (heterozygotycznoœæ obserwowana i oczekiwana, testy: i exact) oraz Carmody ego (test homogennoœci), otrzymane dziêki urzejmoœci Pani mgr P. Wolañskiej-Nowak (Instytut Eksertyz S¹dowych w Krakowie) i Pana dra P. Kozio³a (Katedra Medycyny S¹dowej AM w Lublinie). WYNIKI BADAÑ I ICH OMÓWIENIE Polimorfizm badanych uk³adów rzedstawia tabela I. W oulacji Górnego Œl¹ska w lokus TH01 obserwowano 6 z 8 alleli oisanych w literaturze [5] i 15 fenotyów; w loci TPOX i CSF1PO znaleziono odowiednio: 5 z 8 [4] i 7 z 10 alleli wymienio-

11 Polimorfizm loci tyu STR: TH01, TPOX nych w iœmiennictwie [8, 19]. Obserwowano 11 fenotyów w lokus TPOX i 16 w CSF1PO. Najczêœciej wystêuj¹cym allelem w lokus TH01 w górnoœl¹skiej oulacji jest allel 9.3 (f = 0,3625), najrzadziej 10 (f = 0,0021). Najczêstsze na Górnym Œl¹sku fenotyy: i ojawiaj¹ siê z czêstoœci¹ f = 0,1667, co ozwala stwierdziæ, e s¹ one owszechne. W lokus TPOX w badanej oulacji dominuj¹ allele 8 (f = 0,5556) i 11 (f = 0,2562) oraz fenotyy 8-8 (f = 0,3333) i 8-11 (f = 0,2469). Choæ lokus CSF1PO jest bardziej ró norodne (oisano 10 alleli), to najczêstszymi allelami s¹ 12 (f = ) i 10 (f = 0,3086), zaœ fenotyami (f = 0,2469) i (f = 0,1605). W adnym z badanych loci nie stwierdzono interalleli. Loci: TH01, TPOX i CSF1PO w regionie Górnego Œl¹ska se³niaj¹ rawo równowagi Hardy ego-weinberga, co wykazuj¹ zastosowane testy: i exact liczony metod¹ Monte Carlo i Markov chain (rogram komuterowy TFPGA, tabela I), odobnie jak w innych, oisanych w literaturze oulacjach Polski, Euroy i œwiata [1, 2, 3, 4, 5, 6, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 21, 22]. Porównanie czêstoœci wystêowania alleli w badanych loci w oulacji Górnego Œl¹ska i w innych regionach Polski rzedstawia tabela II. Z tabeli tej wynika, i na oziomie istotnoœci = 0,05 tylko trzy z badanych oulacji Polski maj¹ odobny rozk³ad alleli jak Górny Œl¹sk. S¹ to: Polska ó³nocno-wschodnia [13, 14], Pomorze Kujawy [8] i Dolny Œl¹sk [3]. Pozosta³e olskie oulacje osiadaj¹ odmienne czêstoœci alleli w uk³adzie TH01. Równie Niemcy [6], S³owenia [22] i Hiszania [4] nie wykazuj¹ zgodnoœci z oulacj¹ górnoœl¹sk¹ od wzglêdem rozk³adu alleli w lokus TH01 ( < 0,05 w teœcie Carmody ego rzy orównaniu tych oulacji). Jedynie W³osi [5] maj¹ zbli ony do oulacji Górnego Œl¹ska rozk³ad czêstoœci alleli w TH01 ( = 0,015). Obserwuje siê równie brak jednorodnoœci w rozk³adzie alleli miêdzy oulacj¹ Górnego Œl¹ska a Jaoni¹ czy Zimbabwe ( = 0,000) [2, 11]. Podobny brak homogennoœci w lokus TH01 wykazuj¹ niektóre olskie oulacje regionalne orównywane na zasadzie ka da z ka d¹ i wszystkie jednoczeœnie (tabela III). Analizuj¹c wyniki zastosowanego testu homogennoœci dla lokus TH01, mo na zauwa yæ, e istotne ró nice owoduj¹: odmienne czêstoœci wystêowania trzech blisko siebie le ¹cych alleli: 9, 9.3 i 10 w oulacjach regionalnych [9, 19, 21] oraz odmienna czêstoœæ owszechnych alleli 6 i 9.3 [19, 21]. Trudnoœci w rawid³owym oznaczaniu alleli 9.3 i 10 mog¹ byæ owodem braku homogennoœci miêdzy oulacjami szeroko rozumianej o³udniowej Polski (tabela III). Byæ mo e oisana w literaturze [20] metoda APLP (the amlified roduct length olymorhism) wykorzystuj¹ca zestaw 3 starterów do amlifikacji lokus TH01 i szybkiego, jednoznacznego ró nicowania alleli 9, 9.3 i 10, ozwoli w rzysz³oœci unikn¹æ tego rodzaju rozbie noœci. Allel 10 wystêuje w Polsce o³udniowo-wchodniej [9] z czêstoœci¹ 0,061, zaœ na Górnym Œl¹sku tylko z czêstoœci¹ 0,0021, bli sz¹ ozosta³ym olskim i euroejskim oulacjom [4, 5, 6, 8, 13, 14]. Czêstoœci wystêowania alleli 6 i 9.3 w o³udniowej Polsce [19, 21] s¹ odobne; w ozosta³ych zaœ regionach Polski rzewa a allel 9.3 [3, 8, 9, 13], co mo e równie w³ywaæ na brak jednorodnoœci rozk³adu alleli w lokus TH01 dla oulacji ca³ej Polski (w teœcie homogennoœci Carmody ego orównywano jednoczeœnie wszystkie oulacje Polski tabela III). Dysroorcji w czêstoœci wystêowania alleli 6 i 9.3 w ró nych regionach Polski nie mo na rzyisaæ b³êdnemu fenotyowaniu tych e;

12 92 E. Raczek, K. DroŸdziok, J. Kabiesz oe³nienie b³êdu interretacyjnego jest tu ma³o rawdoodobne. A jednak zaobserwowane niezgodnoœci istotne statystycznie nie mog¹ byæ t³umaczone jedynie odmiennoœci¹ regionaln¹ (suboulacjami) czy ma³¹ liczb¹ badanych osobników. Nale y braæ od uwagê równie b³êdne tyowanie alleli w oarciu o dostêne drabiny alleliczne Promegi (w firmowej drabinie nie wystêuje allel 9.3) czy niedoskona³y system detekcji roduktów amlifikacji. Znamienna jest wysoka zgodnoœæ dwóch oulacji Polski ó³nocnej [12, 17] i Polski o³udniowej [19, 21] badanych w ró nych oœrodkach. Idealn¹ zgodnoœæ obserwowano tak e w loci TPOX i CSF1PO dla oulacji krakowskich [19, 21] (tabela III). Nie stwierdzono natomiast odmiennoœci regionalnej w rozk³adzie czêstoœci alleli w lokus TPOX, znacznie ³atwiejszym w fenotyowaniu i mniej zró nicowanym. W tym lokus oulacja górnoœl¹ska jest homogenna z innymi oulacjami Polski (tabela II) jak równie z oulacjami euroejskimi ( > 0,05) [4, 5, 6, 22]; rzejawia natomiast istotn¹ statystycznie odmiennoœæ od oulacji azjatyckiej i negroidalnej ( = 0,000) [2, 11]. Porównanie wszystkich oulacji olskich miêdzy sob¹ (tabela III) wskazuje jednak na odmienny rozk³ad alleli w lokus TPOX w niektórych z nich. Polska o³udniowo-wschodnia [9] nie jest homogenna z oulacj¹ Pomorza Kujaw [8], Polski o³udniowej [19] i Dolnego Œl¹ska [3]; Dolny Œl¹sk ró ni siê równie rozk³adem alleli w lokus TPOX od Polski o³udniowej [19, 21]. Te odmiennoœci nie owoduj¹ jednak braku jednorodnoœci czêstoœci wystêowania alleli w lokus TPOX w ca³ej Polsce (tabela III). Z kolei w lokus CSF1PO oulacja górnoœl¹ska i ozosta³e zbadane regiony Polski z wyj¹tkiem Polski o³udniowo-wschodniej [9] s¹ wzglêdem siebie jednorodne w rozk³adzie alleli (tabela II). Homogennoœæ wykazuje równie wzglêdem siebie w czêstoœci wystêowania alleli w lokus CSF1PO wiêkszoœæ olskich oulacji (tabela III). Z orównania rozk³adu alleli w tym lokus w badanych oulacjach wynika, e Polska o³udniowo-wschodnia [9] jest regionem o statystycznie istotnej odmiennoœci od Dolnego i Górnego Œl¹ska [3]. Te odmiennoœci nie w³ywaj¹ na jednorodnoœæ rozk³adu alleli w lokus CSF1PO we wszystkich badanych oulacjach Polski (tabela III). Dane statystyczne istotne dla badañ sornego ojcostwa dla trzech badanych loci tyu STR zebrano w tabeli IV. Dane te nie odbiegaj¹ od wyników uzyskanych dla innych regionów Polski [3, 8, 9, 12, 13, 14, 17, 18, 19, 21] i Euroy [4, 5, 6, 22]. ¹czna si³a dyskryminacji (PD) i teoretyczna szansa wykluczenia ojcostwa (MEC), zwana inaczej rzydatnoœci¹ uk³adu (PE), dla badanych loci wynosi odowiednio 0,9973 i 0,8448, co otwierdza wysok¹ rzydatnoœæ tych uk³adów w rozstrzyganiu sraw dotycz¹cych sornego ojcostwa. Wœród 111 takich sraw, w których oznaczano trileks CTT (73) lub tylko TH01 (38), nie sotkano mutacji ani adnych odchyleñ od raw dziedziczenia. Odnotowano natomiast 10 wykluczeñ nies³usznie ozwanych mê - czyzn, wœród których w 8 srawach wyst¹i³o równie wykluczenie ozwanego na odstawie badania uk³adu TH01, TPOX lub CSF1PO. Najczêœciej wy³¹czano nies³usznie ozwanego mê czyznê na odstawie wyniku uzyskanego dla lokus TH01 (w 8 rzyadkach), rzadziej w CSF1PO (w 6) i TPOX (w 3). Te sostrze enia oraz rzedstawiona w tabeli IV charakterystyka rzydatnoœci oszczególnych loci w badaniach sornego ojcostwa zgodnie otwierdzaj¹ najwiêksz¹ rzydatnoœæ trileksu CTT, a z ojedynczych uk³adów TH01.