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Linkage disequilibrium analysis of 67 SNP loci on X chromosome
Forensic Science International: Genetics Supplement Series 3 (2011) e431–e432
Contents lists available at ScienceDirect
Forensic Science International: Genetics Supplement Series journal homepage: www.elsevier.com/locate/FSIGSS
Linkage disequilibrium analysis of 67 SNP loci on X chromosome Li Li *, Shumin Zhao, Yan Liu, Chengtao Li, Yuan Lin, Suhua Zhang Shanghai Key Laboratory of Forensic Medicine, Institute of Forensic Sciences, Ministry of Justice, PR China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 1 August 2011 Received in revised form 7 September 2011 Accepted 15 September 2011
In order to assess the patterns of linkage disequilibrium (LD) about 67 SNP loci on X chromosome, genomic DNA samples extracted from blood fluids from 213 unrelated Chinese Han female individuals were analyzed through multiplex amplification followed by MALDI-TOF MS. LD was analyzed by SNPAnalyzer 2.0. Slight LD were found at two pairs and tight LD were observed at six pairs. From the 67 XSNP loci, 56 informative X-SNPs showing independent inheritance could be chosen out. Both of the combination discrimination power and the cumulative exclusion probability of the 56 markers were above 0.999999. It may be concluded that the panel of 56 X-SNPs loci is valuable in forensic identification. ß 2011 Elsevier Ireland Ltd. All rights reserved.
Keywords: SNP X chromosome Multiple PCR MALID-TOF mass Polymorphism
1. Introduction A panel of informative X-SNPs may be more valuable than autosomal STRs in some deficiency cases, but linkage disequilibrium (LD) should be studied prior to apply the loci to forensic purpose. In the present X-SNP genotyping protocol, 67 SNPs on X chromosome were amplified in four multiplex PCR systems and genotyped by matrix-assisted laser desorption/ionization time-offlight mass spectrometry (MALDI-TOF MS) in Chinese Han female population samples. Hardy–Weinberg equilibrium and linkage disequilibrium were tested and analyzed. Forensic genetic parameters were calculated. 2. Materials and methods 2.1. Sample collection 213 blood samples were collected from unrelated Chinese Han female individuals. DNA was extracted using a BLOOD DNA EXTRACTION kit according to manufacturer’s instructions (Sangon Biotech, Shanghai, China). 2.2. Primer design 67 SNP markers (shown in Table 1) on X chromosome with high minimal allele frequencies (MAF > 0.4) were selected from
* Corresponding author. Tel.: +86 021 52352959; fax: +86 021 52352959. E-mail address: [email protected] (L. Li). 1875-1768/$ – see front matter ß 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.fsigss.2011.09.077
HapMap and NCBI SNP database. The locus to locus space is about 3–20 Mb. 67 groups of primes were designed via MassARRAY Assay Design software (Sequenom Inc.). Each group has three primes, including a pair of PCR primes and a single base extension prime. Primers were synthesized by Sangon Biotech (Shanghai, China). 2.3. SNP typing SNP genotyping was performed using the MassARRAY MALDITOF MS platform (Sequenom Inc.) [1,2]. Briefly, three 17-plex and one 16-plex amplification reactions were processed following standard protocols for iPLEX chemistry. The reaction products were used as templates for the primer extension reactions and then spotted onto the MassARRAY SpectroCHIP. The target plate was inserted into the MALDI-TOF mass spectrometer of MassARRAY compact System and SNP loci was genotyped by MassArray TyperAnalyzer software version 4.0. 2.4. Statistical analysis Hardy–Weinberg equilibrium (HWE) and LD was analyzed by SNPAnalyzer 2.0. The formula for calculation of expected heterozygosity (HET) was introduced by Nei and Roychoudhury [3]. The formula for calculation of discrimination power and exclusion probability were published by Desmarais et al. [4]. 3. Results and discussion Slight LD (r2 > 0.2) were found at two pairs (rs4276834 vs rs5928614 and rs5968332 vs rs6418330) and tight LD (r2 > 0.8)
L. Li et al. / Forensic Science International: Genetics Supplement Series 3 (2011) e431–e432
e432
Table 1 Characters of 67 SNP loci on X chromosome. No.
refSNP
Allele
No.
refSNP
Allele
No.
refSNP
Allele
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23
rs2694717 rs6639989 rs6639398 rs5989571 rs17280621 rs2214174 rs1468160 rs1229078 rs5980274 rs5955927 rs12840669 rs6633608 rs5986750 rs5986751 rs5926442 rs973212 rs1544545 rs6631828 rs4276834 rs5928614 rs5927322 rs6527549 rs183277
A/C G/T A/G A/T A/G A/G A/G A/T C/T C/T C/T A/T A/G C/T C/T C/T A/T C/T A/G A/G A/G C/T G/A
24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46
rs7060326 rs5964206 rs5906919 rs4442270 rs6611148 rs4826623 rs9781645 rs3924423 rs1874111 rs17264553 rs471205 rs7880460 rs2209420 rs7471388 rs5912619 rs2768595 rs5968332 rs6418330 rs10855633 rs5923750 rs1166756 rs2808742 rs7876774
C/T C/G A/G T/C G/T C/T C/T C/T A/T G/T A/G G/T A/C C/G A/G A/T G/T A/G G/T A/G C/G A/G C/T
47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67
rs5920670 rs6620798 rs5917032 rs5916781 rs6418251 rs12849634 rs6646036 rs2519557 rs6649211 rs5956616 rs594031 rs149860 rs5932595 rs2070289 rs5933388 rs5931302 rs203648 rs5908324 rs5954988 rs5905043 rs611711
A/G A/G C/G A/G A/C G/T C/T G/T A/C A/G C/T C/G G/T A/G C/T C/G A/G A/G C/T C/T G/A
were observed at six pairs (rs5986750 vs rs5986751; rs183277 vs rs7060326; rs6611148 vs rs4826623; rs5923750 vs rs1166756; rs6620798 vs rs5917032, rs2519557 vs rs6649211). About the loci observed LD, it is proposed to use haplotype frequencies instead of frequencies of single SNP in calculation of likelihood, or to apply them by preventing the linkage groups. The exact test of 67 X-SNPs except rs12849634 showed no deviation from Hardy–Weinberg equilibrium (p > 0.05). The XSNPs except rs1229078 and rs1544545 were high informative (HET 0.3). As a result, 56 informative X-SNPs showing independent inheritance could be screened out from the 67 XSNP loci. The combination discrimination power of the 56 X-SNPs in Chinese Han female population and male population were 0.999999999999999999999986 and 0.999999999999999980 respectively. The cumulative exclusion probability in duos and in trios were 0.99999974 and 0.999999999987 respectively. It may be concluded that the panel of 56 X-SNPs loci is valuable in forensic identification.
Conflict of interest None. Acknowledgements This work was supported by grants from Shanghai Natural Science Foundation and National Institute Scientific Program (Nos. 08ZR1419800 and GY0805). References [1] W. Pusch, J.H. Wurmbach, H. Thiele, et al., MALDI-TOF mass spectrometry-based SNP genotyping, Pharmacogenomics 3 (2002) 537–548. [2] J. Tost, I.G. Gut, Genotyping single nucleotide polymorphisms by MALDI mass spectrometry in clinical applications, Clin. Biochem. 38 (2005) 335–350. [3] M. Nei, A.K. Roychoudhury, Sampling variances of heterozygosity and genetic distance, Genetics 76 (2) (1974) 379–390. [4] D. Desmarais, Y. Zhong, R. Chakraborty, et al., Development of a highly polymorphic STR marker for identity testing purposes at the human androgen receptor gene (HUMARA), J. Forensic. Sci. 43 (1998) 1046–1049.
Contents lists available at ScienceDirect
Forensic Science International: Genetics Supplement Series journal homepage: www.elsevier.com/locate/FSIGSS
Linkage disequilibrium analysis of 67 SNP loci on X chromosome Li Li *, Shumin Zhao, Yan Liu, Chengtao Li, Yuan Lin, Suhua Zhang Shanghai Key Laboratory of Forensic Medicine, Institute of Forensic Sciences, Ministry of Justice, PR China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 1 August 2011 Received in revised form 7 September 2011 Accepted 15 September 2011
In order to assess the patterns of linkage disequilibrium (LD) about 67 SNP loci on X chromosome, genomic DNA samples extracted from blood fluids from 213 unrelated Chinese Han female individuals were analyzed through multiplex amplification followed by MALDI-TOF MS. LD was analyzed by SNPAnalyzer 2.0. Slight LD were found at two pairs and tight LD were observed at six pairs. From the 67 XSNP loci, 56 informative X-SNPs showing independent inheritance could be chosen out. Both of the combination discrimination power and the cumulative exclusion probability of the 56 markers were above 0.999999. It may be concluded that the panel of 56 X-SNPs loci is valuable in forensic identification. ß 2011 Elsevier Ireland Ltd. All rights reserved.
Keywords: SNP X chromosome Multiple PCR MALID-TOF mass Polymorphism
1. Introduction A panel of informative X-SNPs may be more valuable than autosomal STRs in some deficiency cases, but linkage disequilibrium (LD) should be studied prior to apply the loci to forensic purpose. In the present X-SNP genotyping protocol, 67 SNPs on X chromosome were amplified in four multiplex PCR systems and genotyped by matrix-assisted laser desorption/ionization time-offlight mass spectrometry (MALDI-TOF MS) in Chinese Han female population samples. Hardy–Weinberg equilibrium and linkage disequilibrium were tested and analyzed. Forensic genetic parameters were calculated. 2. Materials and methods 2.1. Sample collection 213 blood samples were collected from unrelated Chinese Han female individuals. DNA was extracted using a BLOOD DNA EXTRACTION kit according to manufacturer’s instructions (Sangon Biotech, Shanghai, China). 2.2. Primer design 67 SNP markers (shown in Table 1) on X chromosome with high minimal allele frequencies (MAF > 0.4) were selected from
* Corresponding author. Tel.: +86 021 52352959; fax: +86 021 52352959. E-mail address: [email protected] (L. Li). 1875-1768/$ – see front matter ß 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.fsigss.2011.09.077
HapMap and NCBI SNP database. The locus to locus space is about 3–20 Mb. 67 groups of primes were designed via MassARRAY Assay Design software (Sequenom Inc.). Each group has three primes, including a pair of PCR primes and a single base extension prime. Primers were synthesized by Sangon Biotech (Shanghai, China). 2.3. SNP typing SNP genotyping was performed using the MassARRAY MALDITOF MS platform (Sequenom Inc.) [1,2]. Briefly, three 17-plex and one 16-plex amplification reactions were processed following standard protocols for iPLEX chemistry. The reaction products were used as templates for the primer extension reactions and then spotted onto the MassARRAY SpectroCHIP. The target plate was inserted into the MALDI-TOF mass spectrometer of MassARRAY compact System and SNP loci was genotyped by MassArray TyperAnalyzer software version 4.0. 2.4. Statistical analysis Hardy–Weinberg equilibrium (HWE) and LD was analyzed by SNPAnalyzer 2.0. The formula for calculation of expected heterozygosity (HET) was introduced by Nei and Roychoudhury [3]. The formula for calculation of discrimination power and exclusion probability were published by Desmarais et al. [4]. 3. Results and discussion Slight LD (r2 > 0.2) were found at two pairs (rs4276834 vs rs5928614 and rs5968332 vs rs6418330) and tight LD (r2 > 0.8)
L. Li et al. / Forensic Science International: Genetics Supplement Series 3 (2011) e431–e432
e432
Table 1 Characters of 67 SNP loci on X chromosome. No.
refSNP
Allele
No.
refSNP
Allele
No.
refSNP
Allele
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23
rs2694717 rs6639989 rs6639398 rs5989571 rs17280621 rs2214174 rs1468160 rs1229078 rs5980274 rs5955927 rs12840669 rs6633608 rs5986750 rs5986751 rs5926442 rs973212 rs1544545 rs6631828 rs4276834 rs5928614 rs5927322 rs6527549 rs183277
A/C G/T A/G A/T A/G A/G A/G A/T C/T C/T C/T A/T A/G C/T C/T C/T A/T C/T A/G A/G A/G C/T G/A
24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46
rs7060326 rs5964206 rs5906919 rs4442270 rs6611148 rs4826623 rs9781645 rs3924423 rs1874111 rs17264553 rs471205 rs7880460 rs2209420 rs7471388 rs5912619 rs2768595 rs5968332 rs6418330 rs10855633 rs5923750 rs1166756 rs2808742 rs7876774
C/T C/G A/G T/C G/T C/T C/T C/T A/T G/T A/G G/T A/C C/G A/G A/T G/T A/G G/T A/G C/G A/G C/T
47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67
rs5920670 rs6620798 rs5917032 rs5916781 rs6418251 rs12849634 rs6646036 rs2519557 rs6649211 rs5956616 rs594031 rs149860 rs5932595 rs2070289 rs5933388 rs5931302 rs203648 rs5908324 rs5954988 rs5905043 rs611711
A/G A/G C/G A/G A/C G/T C/T G/T A/C A/G C/T C/G G/T A/G C/T C/G A/G A/G C/T C/T G/A
were observed at six pairs (rs5986750 vs rs5986751; rs183277 vs rs7060326; rs6611148 vs rs4826623; rs5923750 vs rs1166756; rs6620798 vs rs5917032, rs2519557 vs rs6649211). About the loci observed LD, it is proposed to use haplotype frequencies instead of frequencies of single SNP in calculation of likelihood, or to apply them by preventing the linkage groups. The exact test of 67 X-SNPs except rs12849634 showed no deviation from Hardy–Weinberg equilibrium (p > 0.05). The XSNPs except rs1229078 and rs1544545 were high informative (HET 0.3). As a result, 56 informative X-SNPs showing independent inheritance could be screened out from the 67 XSNP loci. The combination discrimination power of the 56 X-SNPs in Chinese Han female population and male population were 0.999999999999999999999986 and 0.999999999999999980 respectively. The cumulative exclusion probability in duos and in trios were 0.99999974 and 0.999999999987 respectively. It may be concluded that the panel of 56 X-SNPs loci is valuable in forensic identification.
Conflict of interest None. Acknowledgements This work was supported by grants from Shanghai Natural Science Foundation and National Institute Scientific Program (Nos. 08ZR1419800 and GY0805). References [1] W. Pusch, J.H. Wurmbach, H. Thiele, et al., MALDI-TOF mass spectrometry-based SNP genotyping, Pharmacogenomics 3 (2002) 537–548. [2] J. Tost, I.G. Gut, Genotyping single nucleotide polymorphisms by MALDI mass spectrometry in clinical applications, Clin. Biochem. 38 (2005) 335–350. [3] M. Nei, A.K. Roychoudhury, Sampling variances of heterozygosity and genetic distance, Genetics 76 (2) (1974) 379–390. [4] D. Desmarais, Y. Zhong, R. Chakraborty, et al., Development of a highly polymorphic STR marker for identity testing purposes at the human androgen receptor gene (HUMARA), J. Forensic. Sci. 43 (1998) 1046–1049.