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Brief Communication A hypervariable STR polymorphism in the CFI gene: Mutation rate and no linkage disequilibrium with FGA Isao Yuasa a,, Nori Nakayashiki b , Kazuo Umetsu c , Hiroaki Nishimukai d , Aya Matsusue e , Koji Dewa b a Division of Legal Medicine, Faculty of Medicine, Tottori University, Yonago 683-8503, Japan b Department of Legal Medicine, Iwate Medical University School of Medicine, Morioka, Japan c Department of Forensic Medicine, Faculty of Medicine, Yamagata University, Yamagata, Japan d Division of Legal Medicine, Ehime University School of Medicine, Ehime, Japan e Department of Forensic Medicine, Fukuoka University School of Medicine, Fukuoka, Japan article info Article history: Received 18 September 2012 Received in revised form 11 October 2012 Accepted 17 October 2012 Available online 30 November 2012 Keywords: Complement factor I Linkage Mutation rate Short tandem repeat (STR) Variant allele abstract A hypervariable short tandem repeat (STR) polymorphism in intron 7 of the human complement factor I gene (CFI) was investigated to estimate the mutation rate in Japanese samples and to test linkage disequi- librium (LD) with an STR in the fibrinogen alpha chain gene (FGA). The expected heterozygosity and the mutation rate of CFI were estimated to be 0.917 and 0.002, respectively. No LD was observed between CFI and FGA. CFI is a useful supplementary marker for forensic science. Ó 2012 Elsevier Ireland Ltd. All rights reserved. 1. Introduction Short tandem repeats (STRs), or microsatellites, are repeating units of one to six base pairs that are highly polymorphic in human genomes. STR polymorphisms are good markers for individualiza- tion and paternity testing in forensic sciences. For the purposes, commercially available typing kits are generally used. However, additional STRs may be needed in some practical cases. An STR in in- tron 7 of the complement factor I gene (CFI) on chromosome 4q25 is unique because of its high polymorphism and population-specific alleles. The sequence of the repeat structure is classified into two groups, L and H. Common formulae can be given as follows: group L: (TTTC) n C(TTTC) 4 (n = 12–30) group H: (TTTC) p (TTCC) q TTC(TTTC) r C(TTTC) 3 (p + q + r = 37–52). Forty-one alleles and microvariants are observed in four inves- tigated populations and consist of alleles 16–34 of group L and al- leles 40.3–55.3 of group H. The group H alleles were confined in Thai and Japanese populations with frequencies of 0.16 and 0.12, respectively, and were not observed in sub-Saharan African and German populations. Expected heterozygosity ranged from 0.889 in a German population to 0.927 in a Thai population. A family study showed that no mutation was found in 54 German and Jap- anese families with 57 offspring [1]. An STR in the fibrinogen alpha chain gene (FGA) is highly poly- morphic, with about 80 alleles, and can be typed with commercially available amplification kits, such as AmpFlSTR Identifiler (Applied Biosystems, Foster City, CA, USA) and Powerplex 16 System (Prome- ga, Madison, WI, USA). FGA is also assigned to the long arm of chromosome 4, but the detailed location is different between two databases: chromosome 4q28 (http://www.ncbi.nlm.nih.gov/gene/ 2243) and chromosome 4q31.3 (http://www.ensembl.org/index.html). In this study, CFI and FGA in Japanese samples were typed to estimate the mutation rates and to test linkage disequilibrium (LD) between them. 2. Materials and methods For simultaneous typing of CFI and FGA, previous samples [1], 150 Japanese families (including three-generation families) from Iwate, Yamagata, Niigata, Tottori, and Fukuoka prefectures, and several population samples from Tottori prefecture were investi- gated. Genomic DNA was extracted from blood and buccal swabs. This study was approved by the Ethical Committee at the Faculty of Medicine, Tottori University. The amplification of the two STR loci by duplex PCR, and electrophoresis and sequencing of PCR 1344-6223/$ - see front matter Ó 2012 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.legalmed.2012.10.005 Corresponding author. Tel.: +81 859 38 6123; fax: +81 859 38 6120. E-mail address: [email protected] (I. Yuasa). Legal Medicine 15 (2013) 161–163 Contents lists available at SciVerse ScienceDirect Legal Medicine journal homepage: www.elsevier.com/locate/legalmed

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Page 1: A hypervariable STR polymorphism in the CFI gene: Mutation rate and no linkage disequilibrium with FGA

Legal Medicine 15 (2013) 161–163

Contents lists available at SciVerse ScienceDirect

Legal Medicine

journal homepage: www.elsevier .com/locate / legalmed

Brief Communication

A hypervariable STR polymorphism in the CFI gene: Mutation rate and nolinkage disequilibrium with FGA

Isao Yuasa a,⇑, Nori Nakayashiki b, Kazuo Umetsu c, Hiroaki Nishimukai d, Aya Matsusue e, Koji Dewa b

a Division of Legal Medicine, Faculty of Medicine, Tottori University, Yonago 683-8503, Japanb Department of Legal Medicine, Iwate Medical University School of Medicine, Morioka, Japanc Department of Forensic Medicine, Faculty of Medicine, Yamagata University, Yamagata, Japand Division of Legal Medicine, Ehime University School of Medicine, Ehime, Japane Department of Forensic Medicine, Fukuoka University School of Medicine, Fukuoka, Japan

a r t i c l e i n f o

Article history:Received 18 September 2012Received in revised form 11 October 2012Accepted 17 October 2012Available online 30 November 2012

Keywords:Complement factor ILinkageMutation rateShort tandem repeat (STR)Variant allele

1344-6223/$ - see front matter � 2012 Elsevier Irelanhttp://dx.doi.org/10.1016/j.legalmed.2012.10.005

⇑ Corresponding author. Tel.: +81 859 38 6123; faxE-mail address: [email protected] (I. Yuas

a b s t r a c t

A hypervariable short tandem repeat (STR) polymorphism in intron 7 of the human complement factor Igene (CFI) was investigated to estimate the mutation rate in Japanese samples and to test linkage disequi-librium (LD) with an STR in the fibrinogen alpha chain gene (FGA). The expected heterozygosity and themutation rate of CFI were estimated to be 0.917 and 0.002, respectively. No LD was observed between CFIand FGA. CFI is a useful supplementary marker for forensic science.

� 2012 Elsevier Ireland Ltd. All rights reserved.

1. Introduction

Short tandem repeats (STRs), or microsatellites, are repeatingunits of one to six base pairs that are highly polymorphic in humangenomes. STR polymorphisms are good markers for individualiza-tion and paternity testing in forensic sciences. For the purposes,commercially available typing kits are generally used. However,additional STRs may be needed in some practical cases. An STR in in-tron 7 of the complement factor I gene (CFI) on chromosome 4q25 isunique because of its high polymorphism and population-specificalleles. The sequence of the repeat structure is classified into twogroups, L and H. Common formulae can be given as follows:

group L: (TTTC)nC(TTTC)4 (n = 12–30)group H: (TTTC)p(TTCC)qTTC(TTTC)rC(TTTC)3 (p + q + r = 37–52).

Forty-one alleles and microvariants are observed in four inves-tigated populations and consist of alleles 16–34 of group L and al-leles 40.3–55.3 of group H. The group H alleles were confined inThai and Japanese populations with frequencies of 0.16 and 0.12,respectively, and were not observed in sub-Saharan African andGerman populations. Expected heterozygosity ranged from 0.889

d Ltd. All rights reserved.

: +81 859 38 6120.a).

in a German population to 0.927 in a Thai population. A familystudy showed that no mutation was found in 54 German and Jap-anese families with 57 offspring [1].

An STR in the fibrinogen alpha chain gene (FGA) is highly poly-morphic, with about 80 alleles, and can be typed with commerciallyavailable amplification kits, such as AmpFlSTR Identifiler (AppliedBiosystems, Foster City, CA, USA) and Powerplex 16 System (Prome-ga, Madison, WI, USA). FGA is also assigned to the long arm ofchromosome 4, but the detailed location is different between twodatabases: chromosome 4q28 (http://www.ncbi.nlm.nih.gov/gene/2243) and chromosome 4q31.3 (http://www.ensembl.org/index.html).In this study, CFI and FGA in Japanese samples were typed to estimatethe mutation rates and to test linkage disequilibrium (LD) betweenthem.

2. Materials and methods

For simultaneous typing of CFI and FGA, previous samples [1],150 Japanese families (including three-generation families) fromIwate, Yamagata, Niigata, Tottori, and Fukuoka prefectures, andseveral population samples from Tottori prefecture were investi-gated. Genomic DNA was extracted from blood and buccal swabs.This study was approved by the Ethical Committee at the Facultyof Medicine, Tottori University. The amplification of the two STRloci by duplex PCR, and electrophoresis and sequencing of PCR

Page 2: A hypervariable STR polymorphism in the CFI gene: Mutation rate and no linkage disequilibrium with FGA

Fig. 1. Automated electropherogram of CFI and FGA after duplex PCR. (a) CFI, genotype 22–37.3, and (b) FGA, genotype 21–25 observed in a Japanese individual.

Table 1Distribution of allele frequencies for CFI in Japanese samples.

Allele n = 264 n = 372 n = 636

16 0.0038 0.0000 0.001618 0.0038 0.0067 0.005519 0.0114 0.0255 0.019720 0.0208 0.0215 0.021221 0.0947 0.0712 0.0810

162 I. Yuasa et al. / Legal Medicine 15 (2013) 161–163

products were carried out as described previously [1]. The primersfor FGA reported by Urquhart et al. [2] were added to the PCR cock-tails in the concentration of 0.2 lM, and the forward primer waslabeled with NED at the 50-terminus. The mutation rate wascalculated as the number of mutations divided by the number ofallelic transfers from parent to child. The 95% confidence interval(CI) for the mutation rate was derived based on the binomial distri-bution and obtained from a website (http://statpages.org/con-fint.html). Unrelated parent pairs and several population sampleswere used to estimate allele frequencies. Estimation of allele andhaplotype frequencies, and testing of Hardy–Weinberg equilibrium(HWE) and LD were performed using the Arlequin program ver.3.11 [3].

22 0.1345 0.1358 0.135223 0.1326 0.1411 0.137624 0.0871 0.1129 0.102225 0.1080 0.1048 0.106126 0.0795 0.0995 0.091226.2 0.0019 0.0000 0.000827 0.0682 0.0685 0.068427.1 0.0019 0.0000 0.000827.2 0.0057 0.0000 0.002428 0.0436 0.0430 0.043229 0.0436 0.0269 0.033830 0.0152 0.0215 0.018931 0.0114 0.0108 0.011032 0.0114 0.0134 0.012633 0.0038 0.0040 0.003934 0.0019 0.0000 0.0008

37.3 0.0000 0.0013 0.000840.3 0.0019 0.0027 0.002441.3 0.0000 0.0027 0.001642.3 0.0038 0.0013 0.002443.3 0.0038 0.0027 0.003144.3 0.0057 0.0027 0.003945.3 0.0057 0.0040 0.004746.3 0.0114 0.0054 0.007947.3 0.0208 0.0094 0.014248.3 0.0114 0.0094 0.010249.3 0.0133 0.0108 0.011850.3 0.0038 0.0215 0.014251.3 0.0208 0.0054 0.011852.3 0.0057 0.0081 0.007153.3 0.0057 0.0027 0.003954.3 0.0000 0.0013 0.000855.3 0.0019 0.0013 0.0016

NT 35 33 38NH 14 17 17FH 0.1155 0.0927 0.1022

3. Results and discussion

DNA samples were simultaneously typed for CFI and FGA byduplex PCR followed by capillary electrophoresis. Fig. 1 shows anew CFI allele, 37.3, observed in a father and his child. Sequencingshowed the variant had (TTTC)11(TTCC)13TTC(TTTC)8(TTTG)(TTTC)C(TTTC)3. A C > G transversion found in a repeat unit (underlined)has often been observed in the group H alleles with lower sizes (al-leles 40.3–44.3) [1]. This variant was the lowest in size among thegroup H alleles.

In a total of 540 parent–child allele transfers, consisting of 114in the previous report [1] and 426 in the present study, one muta-tion was observed in CFI. A father, mother and child were typed as22–31, 23–26 and 23–30, respectively. Loss of one repeat unit,(TTTC)27 > 26, in the polymorphic repeat region of group L alleleswas observed in paternal meiotic transfer. Group H alleles werefound in 45 children, and one of their parents also had the same al-lele. No mutation was observed in the group H alleles. The muta-tion rate in CFI was estimated to be 0.0019/meiosis (95% CI:0.0000–0.0103). When the German families were excluded, themutation rate in 498 allele transfers was 0.0020/meiosis (95% CI:0.0001–0.0111). No mutation in FGA was observed in this Japanesesample (95% CI: 0.0000–0.0074).

Tables 1 and 2 show allele frequencies at both loci in 636unrelated Japanese individuals as well as in 264 in the previousreport [1] and 372 in the present study. The exact test for HWEshowed that the genotype distribution did not deviate from HWEin every sample case. Thirty-eight CFI alleles, consisting of 21group L and 17 group H alleles, were observed in the 636 individ-

uals investigated. The highest frequency was 0.138 for allele 23.The expected heterozygosity was calculated to be 0.917. The FGAlocus consisted of 18 alleles with the expected heterozygosity of0.861. This value was comparable to that of previous reports on

Page 3: A hypervariable STR polymorphism in the CFI gene: Mutation rate and no linkage disequilibrium with FGA

Table 2Distribution of allele frequencies for FGA in Japanese samples.

Allele n = 264 n = 372 n = 636

17 0.0000 0.0013 0.000818 0.0303 0.0255 0.027519 0.0795 0.0753 0.077020 0.1250 0.1035 0.112421 0.1269 0.1331 0.130521.2 0.0038 0.0000 0.001622 0.1667 0.1962 0.184022.2 0.0000 0.0040 0.002423 0.2159 0.1962 0.204423.2 0.0019 0.0000 0.000824 0.1345 0.1492 0.143124.2 0.0000 0.0027 0.001625 0.0758 0.0780 0.077025.2 0.0019 0.0000 0.000826 0.0303 0.0269 0.028326.2 0.0000 0.0013 0.000827 0.0019 0.0054 0.003928 0.0057 0.0013 0.0031

NT 14 15 18Ho 0.8712 0.8575 0.8632He 0.8635 0.8603 0.8614HWE 0.7303 0.9639 0.9634SD 0.0003 0.0001 0.0001

NT: number of STR alleles observed.Ho: observed heterozygosity.He: expected heterozygosity.HWE: probability value of Hardy–Weinberg equilibrium.SD: standard deviation.

Table 3Exact test of LD between CFI and FGA.

n = 264 n = 372 n = 636

Exact P 0.40515 0.22733 0.05426SD 0.00466 0.00398 0.00224

Exact P: probability value.SD: standard deviation.

Table 1 (continued)

Allele n = 264 n = 372 n = 636

Ho 0.8977 0.8979 0.8978He 0.9205 0.9140 0.9167HWE 0.6048 0.7824 0.8813SD 0.0003 0.0002 0.0001

NT: number of STR alleles observed.NH: number of group H alleles observed.FH: frequency of group H alleles.Ho: observed heterozygosity.He: expected heterozygosity.HWE: probability value of Hardy–Weinberg equilibrium.SD: standard deviation.

I. Yuasa et al. / Legal Medicine 15 (2013) 161–163 163

Japanese (0.862 and 0.856) [4,5]. CFI had higher genetic diversitythan FGA. The exact test for LD showed that there was no signifi-cant LD between the two loci (P > 0.05), as shown in Table 3.

Among STR systems included in the AmpFlSTR Identifiler andPowerplex 16 System, Penta E, D2S1338, FGA and D18S51 werethe most informative for the Japanese population, with their heter-ozygosity ranging from 0.912 to 0.844 [4,5]. The CFI system studied

here was evidently more diverse than the systems mentionedabove and was comparable to ACTBP2 with heterozygosity of0.916 in a Japanese population [6]. ACTBP2 is well-known to havethe highest value of heterozygosity throughout the world.

Lower mutation rates are ideal for use in forensic analyses, andSTRs have mutation rates ranging from 0 to 0.007 [7]; generally,the higher the diversity of STR, the higher the mutation rate ofSTR. A positive exponential correlation between the geometricmean of the number of repeats and the mutation rate has been ob-served [7]. The repeat structure of group L alleles of CFI is very sim-ple and the number of uninterrupted repeats ranges from 12 to 30.In 498 meiotic allele transfers, only one mutation was observed inCFI, with a mutation rate of 0.002. For FGA no mutation was de-tected, suggesting that CFI has somewhat high mutation rate incomparison with FGA. No previous report on the mutation rate ofFGA in Japanese populations was available. In Chinese populationsit was shown to be 0.00217 and 0.0022 in 13,380 and 6,441 alleletransfers, respectively [8,9]. Thus, the mutation rate of CFI may behigher than the estimate (0.002) in this study. ACTBP2, also in-cluded in commercially available amplification kits, is known tohave the highest mutation rate [7]. In a Japanese population itwas reported to be as high as 0.0087 in 230 meioses [10].

It is preferable for STR loci to be located on different chromo-somes. STRs on the same chromosome should be more than50 Mb in physical distance to ensure full recombination and thusindependent inheritance. CFI and FGA are about 45 Mb or 40 cMdistant from each other (http://mathgen.stats.ox.ac.uk/Recombina-tion.html). The exact test for LD showed that the two STRs can betreated as genetically independent markers. Finally, CFI is a usefulsupplementary marker for forensic science because of high diver-sity, moderate mutation rate and no LD with FGA.

Acknowledgements

This study was supported in part by a Grant-in-Aid for ScientificResearch (20590678 and 23590849 to I.Y.) from the Japan Societyfor the Promotion of Science.

References

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