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Genetics and Molecular Biology

Print version ISSN 1415-4757On-line version ISSN 1678-4685

Genet. Mol. Biol. vol.31 no.3 São Paulo  2008 



Population and mutation analysis of Y-STR loci in a sample from the city of São Paulo (Brazil)



José A. Soares-VieiraI; Ana E.C. BillerbeckII; Edna S.M. IwamuraI; Berenice B. MendoncaII; Leonor GusmãoIII; Paulo A. OttoIV

IDepartamento de Medicina Legal, Faculdade de Medicina, Universidade de São Paulo, São Paulo, SP, Brazil
IILaboratório de Hormônios e Genética Molecular, 1ª Clínica Médica, Hospital das Clínicas, Faculdade de Medicina, Universidade de São Paulo, São Paulo, SP, Brazil
IIIInstituto de Patologia e Imunologia Molecular, Universidade do Porto, Porto, Portugal
IVDepartamento de Genética e Biologia Evolutiva, Instituto de Biociências, Universidade de São Paulo, São Paulo, SP, Brazil

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The haplotypes of seven Y-chromosome STR loci (DYS19, DYS389I, DYS389II, DYS390, DYS391, DYS392, and DYS393) were determined in a sample of 634 healthy Brazilian males (190 adult individuals and 222 father-son pairs). The 412 adults were unrelated, and the 222 father-son pairs had their biological relationship confirmed using autosomal STRs (LR > 10,000). Among the 412 adults, a total of 264 different 7-loci haplotypes were identified, 210 of which were unique. The most frequent haplotype was detected in 31 instances, occurring with a frequency of 7.52%. The haplotype diversity index was calculated as 98.83%. Upon transmission of the 1,554 alleles, in 222 father-son pairs, six mutations were observed, with an average overall rate of 3.86 x 10-3 per locus. A haplotype with a duplicated DYS389I locus, and another with duplicated DYS389I, DYS389II, and DYS439 loci were detected in both fathers and their respective sons.

Key words: Y-STR population data, São Paulo (Brazil), mutation rates, duplications.



Y-chromosome STR typing has become an important tool in forensic analysis (Betz et al., 2001; Sibille et al., 2002; Cerri et al., 2003; Shewale et al., 2003; Shewale et al., 2004; Delfin et al., 2005; Johnson et al., 2005). Recently, the DNA Commission of the International Society of Forensic Genetics (ISFG) has published guidelines and recomendations concerning the use of Y-STRs polymorphisms in human identification and kinship analysis (Gusmão et al., 2005). According to a recent Brazilian government census (IBGE), 54% of Brazilians were self-declared as white, 38% as mixed (mulatto), and 6% as black; 2% were classified in other categories that include Orientals and Amerindians, with striking regional differences. For instance, mixed tri-hybrid types are overwhelmingly predominant (almost 100%) in some parts of the northeastern region, whereas whites vastly predominate in the southern states (almost 100% in some inner regions of the states of Santa Catarina and Rio Grande do Sul). Several studies performed in different population samples from Brazil (Costa et al., 2002; Grattapaglia et al., 2005; Cainé et al., 2005;; Carvalho-Silva et al., 2006; Silva et al., 2006; Domingues et al., 2007; Palha et al., 2007) have shown, however, that in spite of this racial melting pot, genes carried on the Y chromosome are almost exclusively of European origin (Iberian, Mediterranean, and Central-European), while analyses of mtDNA variability in Brazilian samples revealed that about 60% of the maternal lineages are Amerindian and African (Carvalho-Silva et al., 2006). Therefore, regardless of this intense gene flow and high degree of genetic heterogeneity, Y chromosome polymorphisms in Brazilian males have a distribution typical of a mixed European population. This paper presents data on 7 Y-STR loci DYS19, DYS389I, DYS389II, DYS390, DYS391, DYS392, and DYS393 in a Brazilian mixed population sample from the city of São Paulo.

Whole blood samples were collected from 634 healthy Brazilian males (190 adult individuals and 222 pairs of fathers and respective sons), under written informed consent. The 412 adults were unrelated and the 222 father-son pairs had their biological relationship confirmed by paternity index values larger than 10,000 obtained by means of autosomal STRs typing. DNA was extracted from 5 mL of peripheral blood by a salting-out procedure (Miller et al., 1988), and quantified by spectrometry (Ultrospec III, Pharmacia, Piscataway, NJ, USA). The amplification of DYS19, DYS389I, DYS389II, DYS390, DYS391, DYS392 and DYS393 loci was performed according to Kayser et al. (1997), in two multiplex reactions, one triplex (DYS391,DYS392, DYS393), and one tetraplex (DYS19, DYS389I, DYS389II and DYS390). One primer of each pair was labeled with a fluorescent dye. In a final volume of 25 µL, 50 ng of genomic DNA was mixed with 200 µM of dNTP, 2.0 mM MgCl2, 2.5 U of Taq polymerase (Amersham Biosciences, Piscataway, NJ, USA), 2.5 µL of the10X reaction buffer provided by the manufacturer, and with the forward and reverse primers. In the triplex reaction, the concentrations of primers were 7.0 pmol for DYS391, 8.5 pmol for DYS392 and 3.0 pmol for DYS393; in the tetraplex reaction, 7.0 pmol for DYS19, 6.0 pmol for DYS389I/II and 4.0 pmol for DYS390. The samples were subjected to 30 cycles of amplification in a 9700 thermal cycler (Applied Biosystems, Foster City, CA). The amplification conditions were 94 °C, 5 min; 35 cycles of 94 °C 1 min, 55 °C 1 min, and 72 °C 1 min; followed by 72 °C 30 min, and 12 °C until the samples were removed from the thermal cycler. Fragment size analysis was performed using the GeneScan 2.1 software. Two microliters of the amplification products were mixed with 24 µL of Hi-Di Formamide (Applied Biosystems), 1 µL of the size standard TAMRA-350 (triplex reaction) or TAMRA-500 (tetraplex reaction) and subjected to capillary electrophoresis on the ABI 310 Genetic Analyzer (Applied Biosystems) using POP-4 (performance optimized polymer), filter set C and an injection time of 5 s. The electrophoresis time was 24 min for the triplex reaction and 28 min for the tetraplex reaction. Four samples were reanalyzed using the AmpFlSTR YFiler kit (Applied Biosystems) as recommended by the manufacturer. Since Y-STRs are haploid, allele and haplotype frequencies, as well as linkage disequilibrium values between pairs of genes at different loci and mutation rates per locus, were estimated by direct counting methods, using computer programs prepared by us. The significance of association measurements [linkage disequilibrium values (ldv) estimated between all possible pairs of alleles belonging to two out of the seven loci here studied] was verified through Fisher exact tests performed on 2x2 contingency tables. All the methods here mentioned are detailed in standard text-books on statistical and population genetic methodology (Zar, 1999; Weir, 2001; Sham, 2002).

Table 1 lists the observed relative frequencies of different Y-STR alleles segregating in each of the seven loci. Table S1 (Supplementary Material) lists the observed absolute and relative frequencies of the 7-loci haplotypes among the 412 unrelated adult subjects.



A total of 264 different haplotypes were identified, 210 of which were unique. The most frequent haplotype (DYS19 14/DYS389I 13/DYS389II 29/DYS390 24/DYS391 11/DYS392 13/DYS393 13) was found in 31 instances (7.52%). The second most frequent haplotype (14/13/29/24/10/13/13), which differed from the previous haplotype by only a single DYS391 repeat, was shared by 14 individuals (3.39%).

Table 2 lists the estimates of the diversity index obtained for each of the 7 Y-STR loci, the average figure calculated for these 7 loci and for the set of 7-loci haplotypes, together with their corresponding standard errors and approximate 95% confidence intervals. While diversity indices for isolated loci ranged from 0.51 to 0.70 with an overall average value of 0.60, the 7-loci haplotype diversity index was of the order of 0.99, as expected, since 210/264 (79.5%) of all haplotypes were unique, each occurring with a frequency of about 0.002.



Table 3 lists the results of association tests performed between the genes of possible pairs of Y-STR loci. Since the number of different statistical tests performed was 859, the critical alpha rejection level (for testing the null hypothesis of ldv = 0) was adjusted following Bonferroni's method, giving a corrected alpha critical value of 0.00006. Therefore, Table 3 lists only the pairs of linked Y-STR loci (out of the 859 tested for linkage disequilibrium) with linkage disequilibrium values [D(i,j)] significantly different from zero at the level of 6 x 10-5 or less. As expected, many (if not most) of these very significant values occurred preferentially between pairs of contiguous loci.



In forensic cases, in which multiple male aggressors are involved, the autosomal STR profiles often provide inconclusive results. Y-STR markers are being increasingly used as potential tools for distinguishing low levels of male DNA in the presence of excess female DNA, which occurs in many sexual assault samples. Due to the haploid nature of Y-STRs, in cases where multiple males are contributors, the number of donors can be estimated by the presence of additional alleles in a Y chromosome profile, usually being interpreted as an admixture of more than one contributor. The most commonly used Y-STR markers are single-copy loci, with the exception of the DYS385 locus. However, many regions of the Y-chromosome are duplicated or even triplicated in some individuals and this fact can thus complicate potential mixture interpretation (Kayser et al., 2000; Bosch and Jobling, 2003; Çakir et al., 2004; Kurihara et al., 2004; Butler et al., 2005; Diederiche et al., 2005;). The precise estimation of the frequency of duplicated mutated Y-STR alleles is thus very important in forensic genetic analyses, because the presence of multiple peaks can be misinterpreted as mixed profiles (Diederiche et al., 2005). In the present study, one sample had a 7 Y-STR haplotype with double peaks at the DYS389I locus, and another presented a 7 Y-STR haplotype with double peaks at DYS389I and DYS389II. The analysis of these two samples was increased to 16 markers, using the AmpF/STR Yfiler Kit, and a double peak was also found in the locus DYS439 of the second sample. Each one of the two pairs of father and son had the same haplotype (Table S2).

The set of 7 Y-STR loci was typed in 222 father-son pairs. Upon 1,554 allele transmissions, six de novo mutations were observed, one mutation had occurred at the DYS392 locus (14 to 13), two mutations at the DYS390 locus (24 to 23; 22 to 24), and three mutations took place at the DYS391 locus (12 to 10; 12 to 11; 11 to 10) (Table 4). Except for two cases (DYS390 and DYS391), all were single-step mutations, and only a single mutation occurred during each father-son transmission.




This work was partially supported by FAPESP and LIM-HC-FMUSP, Brazil. IPATIMUP is partially supported by Fundação para a Ciência e a Tecnologia, through POCI (Programa Operacional Ciência e Inovação 2010).



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Internet Resources

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Send correspondence to:
José Arnaldo Soares-Vieira
Departamento de Medicina Legal, Faculdade de Medicina, Universidade de São Paulo
Rua Teodoro Sampaio 115
05405-000, São Paulo, SP, Brazil

Received: March 14, 2008; Accepted: June 9, 2008.



Associate Editor: Francisco Mauro Salzano



Supplementary Material

The following online material is available for this article:

- Table S1. Observed absolute and relative frequencies of STR-Y 7-loci haplotypes.



- Table S2. Y-STR haplotype profiles showing the presence of additional alleles.


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