DYS464: quatro cópias (a, b, c, d)
2.6 Estudos do Cromossomo Y em Populações Brasileiras
Portugal foi a principal fonte de imigrantes europeus que chegaram ao Brasil entre 1500 e 1808. Neste período cerca de 500.000 imigrantes
l. 2001). Da etad
rasil é resultante da intensa miscigenação corrida principalmente entre as linhagens ameríndias, européias e fricanas (Pena et al. 2000).
Estudos do cromossomo Y têm demonstrado que a origem paterna da população brasileira é predominantemente européia. De acordo com Santos et al. (2003) a diversidade de haplótipos de microssatélites do Y da população da Bahia é semelhante à de Portugal e significativamente maior que a de populações do noroeste da África, região de origem da maioria dos africanos transportados para a Bahia. Na população do Rio de Janeiro dentre 129 haplótipos foram encontrados sete haplótipos que provavelmente seriam de origem banto (Góes et al. 2004). O exame de polimorfismos da NRY em 200 brasileiros auto definidos como brancos, de várias regiões geográficas, demonstrou que a grande maioria dos cromossomos Y (97,5 %) é de origem européia, 2,5 % de origem africana (sul da África) e nenhum de origem ameríndia (Carvalho-Silva et al. 2001).
Estudos comparativos têm demonstrado que a variação haplotípica entre as populações das cinco regiões geopolíticas do Brasil não é significativa (Santos et al. 2003; Grattapaglia et al. 2004).
portugueses chegaram ao Brasil sendo a maioria homens (IBGE 2000). Os primeiros brasileiros foram resultado do casamento entre homens portugueses e mulheres ameríndias (Carvalho-Silva et a
m e do século 16 até o século 19 estima-se que 4.000.000 de
escravos africanos foram trazidos para o Brasil. No período que vai de 1820 a 1975, 70 % dos mais de cinco milhões de imigrantes europeus que chegaram oficialmente no Brasil eram portugueses e italianos (IBGE 2000). A população do B
o a
Os microssat ma de pesquisas alizadas no mundo todo e isto é confirmado pelo grande número de dem
a gen
élites do cromossomo Y têm sido o te re
trabalhos publicados bem como de populações estudadas. Este fato onstra a importância destes marcadores para a ciência forense e para
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ceill D and Erlich SD (2001) Replication slip
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Diversity and Mutation Analysis of Minimal Haplotype Y-
Microsatellites Loci Plus DYS447, DYS458 and DYS464 in Alagoas, Northeastern Brazil
Genética, Centro de Ciências Bioilógicas, Universidade
ing author: Luiz M
Dalmo A. de Azevedo,1 M.Sc.; Luiz A.F.da Silva,2 Ph.D.; Adriana B.G.
Barbosa.1 M.Sc.; and Luiz Mauricio-da-Silva,3 Ph.D.
1 Departamento de
Federal de Pernambuco.
2 Laboratório de DNA Forense, Departamento de Biologia, Universidade
Federal de Alagoas.
3 Laboratório de Genética Molecular Humana, Departamento de Genética,
Universidade Federal de Pernambuco
* This study was supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico – CNPq
Correspond
auricio da Silva Dept. de Genética – CCB
Universidade Federal de Pernambuco – Cidade Universitária 50.000-000 Recife, PE, Brazil
Population: A total of 510 males representing the State of Alagoas,
Northeast Brazil, comprised 255 unrelated individuals from which was also typed the respective son.
Keywords: forensic science, DNA typing, Y-microsatellites, minimal Y- chrom
The S
silver stained. Home made allelic ladders were used for allele designation. osome haplotype, DYS447, DYS458, DYS464, population data, Alagoas, Brazil.
tate of Alagoas is located in Northeast Brazil and the population of this region can be characterized by intense admixture of three ethnical groups, Caucasian, African and Native Amerindians (1, 2). Therefore the Alagoas population form one of the most heterogeneous populations of the world. No data was published on Y chromosome microsatellites for Alagoas population, to date. In this work, we studied the nine microsatellites loci of the minimal haplotype (DYS19, DYS385, DYS389I and II, DYS390, DYS391, DYS392 and DYS393) plus DYS447, DYS458 and DYS464.
Blood samples from 255 unrelated males and his sons, in a total of 510 individuals were provided from the paternity caseworks of the whole State of Alagoas, with personal consent. DNA was extracted through chelex method (10). The amplifications were performed in four PCR reactions: 1) DYS19, DYS389I and II; 2) DYS392, DYS393; 3) DYS385, DYS390, DYS391; 3) DYS447, DYS458, DYS464. Each PCR reaction used 20 ng of genomic DNA, in a total reaction volume of 25 µl. Published primers were used: DYS19, DYS390, DYS392 and DYS393, Kayser et al. (3); DYS385, Schneider et al. (4); DYS389I and II, Schultes et al. (5); DYS391, Wiegand and Kleiber (6); DYS447, Butler et al. (7); DYS458, Schoske et al. (8); and DYS464, Redd et al. (9). PCR cycling protocol was: 95° C, 2min; 28 cycles: 94° C, 1 min.; 56° C, 2 min.; 72° C, 30 min.
For the minimal haplotype, loci allele calibration was made by using the 9948 male DNA control (Promega Corporation) and by typing four samples
with P d
DYS464 allele designation were confirmed by sequencing at least two locus.
and h
rate was estimated based on allele transmitions. frequ
represented only once. In the same sample for minimal haplotype, the mo
frequ
individuals sample from the five geopolitical regions in Brazil. The gene an
DYS4 diver
The m DYS458 with a gene diversity of
0.7
the D l
ha amon
in DYS458. All mutations were confirmed by reanalysis and, for DYS390 an
occur
was o ther/son pair. The
ov confid the 9
the haplotype composed by this markers set is highly informative and
owerPlex Y® kit (Promega Corporation). For DYS447, DYS458 an
alleles of each
Analysis of data was carried out using Arlequin ver. 2000 (11). Gene aplotype diversities were estimated according to Nei (12). Mutation A total of 230 haplotypes were observed, where the three most ent ones occurred three times, 19 occurred two times and 207 were st frequent haplotype was observed 16 times, being the same most ent haplotype observed by Grattapaglia et al. (13), in a 198 d haplotype diversity are shown in table 1. The multi copy markers
64 and DYS385 were the most polymorphic ones, with a gene sity respectively of 0.9398 ± 0.0080 and 0.8930 ± 0.0147 (table 1).
ost polymorphic single copy was
756 ± 0.0129. The haplotype diversity increased considerably when YS447, DYS458 and DYS464 loci was added to the minima plotype loci (see table 1). Eight mutations were observed (table 2)
g 3,825 allele transmitions, three in DYS390 and in DYS464 and two d DYS458, by DNA sequence analysis, and they were found to have
red inside the repetitive sequence structure. Only one step mutation bserved and find in only one locus for the same fa
erall mutation rate estimate, across the 15 loci, was 2.09 × 10-3 (95%
ence interval (CI) 9.03 × 10-4 to 4.11 × 10-3). This value is inside
dis in for
at http://www.mhn.ufal.br Refe
1. Donadi EA. Population
2. The
6. eduction of STR
amplicons using redesigned primers. Int J Legal Med 2001; 114: 285-87.
7. Butler MB, Schoske R, Vallone PM, Kline MC, Redd AJ, Hammer MF. A novel multiplex for simultaneous amplification of 20 Y chromosome STR markers. Forensic Sci Int 2002; 129: 10-24.
8. Schoske R, Vallone PM, Kline MC, Redman JW, Butler MB. High- throughput Y-STR typing of U.S. populations with 27 regions of the Y chrosome using two multiplex PCR assays. Forensic Sci Int 2004; 139: 107-21.
criminative for male lineages in Alagoas, with potential for application ensic casework.
The complete dataset can be accessed rences
Mauricio-da-Silva L, Silva RS, Dellalibera E,
genetics of HPRTB, F13B, and LPL in Pernambuco, Northeast Brazil. J Forensic Sci 2000; 45: 684-86.
Carvalho-Silva DR, Santos FR, Rocha J, Pena SDJ. phylogeography of Brazilian Y-chromosome lineages. Am J Hum Gen 2001; 68: 281-86.
3. Kayser M, Caglià A, Corach D, Fretwell N, Gehrig C, Graziosi G et al. Evaluation of Y-chromosomal STRs: a multicenter study. Int J Legal Med 1997; 110: 125-33.
4. Schneider PM, Meuser S, Wayawuth W, Seo Y, Rittner C. Tandem repeat structure of the duplicated Y-chromosomal STR locus DYS385 and frequency studies in the German and three Asian populations. Forensic Sci Int 1998; 97: 61-70.
5. Schultes T, Hummel S, Herrmann B. Amplification of Y-chromosomal STRs from ancient skeletal material. Hum Gen 1999; 104: 164-66. Wiegand P, Kleiber M. Less is more – length r
9. Redd AJ, Agellon AB, Kearney VA, Contreras VA, Karafet T, Park H et al. Forensic value of 14 novel STRs on the human Y chromosome. Forensic Sci Int 2002; 130: 97-111.
10. Walsh PS, Metzger DA, Higuchi R. CHELEX ® 100 as a medium for
simple extraction of DNA for PCR-based typing from forensic material. Bio Techniques 1991; 10: 506-13.
2.000: a cs data analysis. Genetics and
eneva, Switzerland, 2000.
M. lar onary genetics. New York: Columbia
rsi 19
attapa Kal , G e ,
res hro STR haplotype diversity in Brazilian
ulati ens 2005; 149: 99-107.
ser er an M, Henke L, Henke J, Brauer S et al.
act nd y of germline mutations at microsatellite
fro hu chromosome, as revealed by direct
-88. ACS, Carvalho EF, Gomes I, Silva DA, Gil EHF, Amorin A et al. Population and mutation analysis of 17 Y-STR loci from Rio de Janeiro (Brazil). Int J Legal Med 2004; 119: 70-76.
11. Schneider S, Roessli D, Excoffier L. Arlequin version software for population geneti
Biometry Laboratory. University of G
12. Nei Molecu evoluti
Unive ty Press, 87.
13. Gr glia D, upniek S uimarães CS, Rib iro MA, Diener PS
Soa CN. Y-c mosome
pop ons. For ic Sci Int
14. Kay M, Roew L, Hedm
Char eristics a frequenc
loci m the man Y
observation in father/son pairs. Am J Hum Gen 2000; 66: 1580 15. Góes
Table 1 – Gene diversities for each locus and haplotypic diversity for minimal haplotype locy and for all 15 loci haplotype.
G.D. = gene diversity; H.D. = haplotypic diversity; s.d. = standard deviation. Marker G. D. s.d. H.D. (minimal haplotype loci) H. D. (all 15 loci) DYS464 0.9398 0.0080 DYS385 0.8930 0.0147 YS389II 0.6552 DYS19 0.6325 0.0266 0.0262 YS392 0.6060 0.0199 0.0146 YS389I 0.4883 0.0311 DYS393 0.4848 0.0335 0.9939 ± 0.0019 0.9991 ± 0.0005 DYS458 0.7756 0.0129 DYS447 0.6845 0.0227 D 0.0224 DYS390 0.6243 D DYS391 0.5770 D
Table 2 – Father/son muta he total number of allele ansmitions were 255 for DYS390 and DYS458, for DYS464 were 1020.
oci Father allele Son allele
tions observed. T tr L DYS390 24 23 DY DY 24 23 23 24 S458 15 14 19 18 S64 12-16 12-15-16 13-16 13-16-17 14-15-16-18 14-15-16-17
5
Mesmo sendo intensamente miscigenada a população de Alagoas
elevado poder de discriminação de linhagens paternas.
A análise de mutações demonstrou que na população de Alagoas a taxa de mutação está de acordo com as taxas de mutação detectadas em outras populações.
.
.
CC
OONNCCLLUUSSÕÕEESSapresentou variabilidade genética, baseada nas diversidades gênica e haplotípica, semelhante á de outras populações do Brasil bem como a de populações dos Estados Unidos, da Europa e da África.
O haplótipo mais comum da população de Alagoas coincide com o haplótipo mais comum das populações do sul de Portugal, da Ligúria (Itália) e de Madri (Espanha), países de onde veio a maioria dos colonizadores europeus para o Brasil. Nenhuma ocorrência foi detectada na população de Alagoas para os haplótipos mais comuns das populações Yanomami, Banto e de Moçambique. Estes fatos favorecem a hipótese de que na formação da população de Alagoas o principal contribuidor do sexo masculino foi o colonizador europeu. A combinação do haplótipo mínimo com os locos DYS447, DYS458 e
6
6..AA
BBSSTTRRAACCTThe use of microsatellites localized in the non recombinant region of Y hromosome (NRY) is widespread in forensic and population studies. In is work the polymorphism and mutation rate analysis was made for 15 Y hromosome microsatellites (minimal haplotype plus DYS447, DYS458 and YS464) in an Alagoas population sample, composed by 255 father/son airs. A total of 230 different haplotypes was observed, of wich 207 curred only once. Through fathers and sons analysis were detected utations in eight of the 3,825 analysed allelic transmitions, with a mean
utation rate estimated of 2.092 × 10-3. The most common minimal
aplotype occurred 16 times it and is also the most common haplotype in aucasians from Rio de Janeiro and São Paulo populations and in
uropean populations of South Portugal, Madri (Spain) and Ligury (Italy). The most common haplotype of Yanomami, Bantu and Moçambique populations was not found in the Alagoas population.
K
KEEYYWWOORRDDSS: forensic science, human identification, microsatellites, Y :
chromosome haplotypes, DYS447, DYS458, DYS464, Alagoas, Brasil. T c th c D p o m m h c E
7
7
7..11..AA
NNEEXXOO11
I
7.1.1 Detalhamento das metodologias utilizadas 7.1.1.1 Obtenção da amostra
am analisados 510 indivíduos do sexo masculino correspondendo s pai/filho com paternidade confirmada através de marcadores índice de paternidade ≥ .000 (probabilidade ≥ 99,9 esta amostra foram analisados indivíduos provenientes de 42
o de Alagoas. Na tabela 1 estão listados estes mero de indivíduos provenientes de cada um.
ção do DNA
foi obtido a partir de sangue total colhido por punção digital
e 30 µl por indivíduo. O sangu oi colocado em microtubos
10 µl de EDTA 100 mM para impedir a coagulação. DNA foi feita pelo método de chelex (Walsh et al. 1991) ificações sendo descritas a seguir. A cionar aos 30 µl de sangue
estilada e esterilizada e após gitação deixar em repouso inutos à temperatura ambiente para hemólise. A seguir
por 2 minutos e descartar o sobrenadante.