Int J Legal Med
DOI 10.1007/s00414-009-0369-0

SHORT COMMUNICATION

A hypervariable STR polymorphism in the complement
factor I (CFI) gene: Asian-specific alleles
Isao Yuasa & Yoshito Irizawa & Hiroaki Nishimukai &Yasuo Fukumori & Kazuo Umetsu & Nori Nakayashiki &Naruya Saitou & Lotte Henke & Jürgen Henke

Received: 29 January 2009 / Accepted: 8 July 2009
# Springer-Verlag 2009

Abstract In this study, a short tandem repeat (STR)
polymorphism in intron 7 of the human complement factor I(CFI) gene was studied in 637 DNA samples obtained fromAfrican, German, Thai, and Japanese populations and German
and Japanese families. A total of 41 alleles were observed andclassified into two groups, L and H, based on size differences.
Group H, which consisted of 16 alleles, was observed only inThai and Japanese populations at frequencies of 0.162 and0.116, respectively, and was strongly associated with c.1217A
in exon 11 (CFI*Ah). The heterozygosity values ranged from
0.89 in German to 0.93 in Thai populations. This STR would
be a useful supplementary marker for forensic individualization.
Keywords Asian-specific . Complement factor I .
Microsatellite . Population study . Short tandem repeat

Introduction
Short tandem repeat (STR) markers are capable of possessing
a high degree of polymorphism and are the most effective
markers for forensic individualization. Population-specific
STR alleles or regionally private STR alleles with a frequency
above 0.02 have sometimes been detected on autosomal
chromosomes. However, no allele with a frequency above
0.13 has been found except for allele 9RA in D9S1120,
which is observed at an average frequency of 0.36 in
Native Americans [1, 2].
The human complement factor I (CFI) gene (GenBank
GeneID: 3426) spans 63 kb on chromosome 4q25 andconsists of 13 exons encoding a 583-amino acid polypeptide
as an unprocessed precursor. Recently, we described Japanese,

Electronic supplementary material The online version of this article(doi:10.1007/s00414-009-0369-0) contains supplementary material,
which is available to authorized users.
I. Yuasa (*) : Y. Irizawa
Division of Legal Medicine, Faculty of Medicine,
Tottori University,
Yonago 683-8503, Japane-mail: yuasai@med.tottori-u.ac.jp
H. Nishimukai
Division of Legal Medicine,
Ehime University School of Medicine,
Ehime, Japan

N. Nakayashiki
Department of Legal Medicine,
Iwate Medical University School of Medicine,Morioka, Japan

N. Saitou
Division of Population Genetics, National Institute of Genetics,
Mishima, JapanY. Fukumori
Osaka Red Cross Blood Center,
Osaka, JapanK. Umetsu
Department of Experimental and Forensic Pathology,
Faculty of Medicine, Yamagata University,
Yamagata, Japan

L. Henke : J. Henke
Institut für Blutgruppenforschung,
Cologne, Germany

Int J Legal Med

African, and East Asian-specific nonsynonymous singlenucleotide polymorphisms (SNPs) in exons 4 (c.A603C), 7(c.G898A), and 11 (c.G1217A) [3]. An STR with 27
repeats of the tetranucleotide polypyrimidine TTTC unit
exists in intron 7 of this gene. The nucleotide sequenceshows an insertion/deletion polymorphism due to thepresence or absence of (TTTC)4, which is registered as
refSNP ID rs56875356 (GenBank Entrez SNP) without
frequency data. In this study, we scrutinized this CFI STRpolymorphism with respect to its repeat structure, allele
distribution, and genetic association with the SNPs andpresent evidence for the hypervariability of the CFI STR
with Asian-specific alleles.

Materials and methods
DNA samples
DNA samples were obtained from 68 sub-Saharan African(Nigerian and Ghanaian), 92 German, 114 Thai, and 198
Japanese individuals. Most of these samples were from thesame set as those used in a previous study [3]. For family
studies, 21 German trios and 33 Japanese families with 36
offsprings were investigated. This study was approved by the
Ethical Committee at the Faculty of Medicine, Tottori
University. Nonhuman primate DNA samples were also
obtained from three chimpanzees (Pan troglodytes), a gorilla(Gorilla gorilla), and a Japanese monkey (Macaca fuscata).
SNP typing
The five SNPs in the CFI gene were typed by a multiplex
polymerase chain reaction (PCR) method as described
previously [3].
PCR
PCR products containing the STR were amplified with thefollowing primers: forward primer CFI-i7F: 5′-AACGTCAAAGTTCATGATCCTC-3′ and reverse primer CFIFig. 1 The GenBank sequence
for intron 7 of the CFI gene. The
primers are underlined and the
STR, which consists of 27
repeats of TTTC motif, is shaded in gray. The nucleotide
numbering was defined according to the genomic sequence(NT_000004.10). The adenineof the translation initiation
codon ATG corresponds tonucleotide 15

i7R3: 5′-AGCCTGGGCTACAAGACC-3′ (Fig. 1). The
forward primer was labeled with VIC at the 5’ terminal.
PCR was carried out in a volume of 10µL containing
1 ng genomic DNA, 0.4µM of each primer, 200µM ofeach dNTP, and 0.25 U HotStarTaq Plus polymerase(Qiagen, Hilden, Germany). The cycle conditions wereas follows: 95°C for 5 min, followed by 30 cycles of
94°C for 30 s, 56°C for 30 s, 72°C for 40 s, and a final
extension of 90 min at 72°C.
Typing of STR
Of the PCR products or allelic ladder, 1µL was added
to 9µL of HiDi-formamide containing GeneScan 500
LIZ size standard (Applied Biosystems, Foster City,
CA, USA), and aliquots were separated by electrophoresis on a 3130xl Genetic Analyzer (Applied Biosystems) with a 36 cm array and POP-7 polymer. The
allelic ladder was made in-house by mixing PCR
products with different genotypes. The sample run data
were analyzed together with the allelic ladder andpositive and negative controls using GeneMapper
Software Version 3.5 (Applied Biosystems). Alleles
were designated according to the recommendations
from the DNA Commission of the International Society
for Forensic Genetics. [4].
Sequencing
The PCR products for sequencing were prepared withsequencing primers under the abovementioned conditions
except the reaction volume was 50µL. The primers usedwere CFI-i7F5: 5′-GTAAAATGACTCATATCCC
CAGTTC-3′ and CFI-i7R2: 5′-GGCAGAGAATTGCTTAAACCC-3′ (Fig. 1). In heterozygotes, each band was
excised from a denaturing gel and subjected to reamplification with the same primer set. The products were
purified using Qiaquick PCR Purification Spin Columns(Qiagen). Sequence reactions were obtained using a
BigDye Terminator v3.1 Cycle Sequencing Kit (Applied
Biosystems).

Int J Legal Med

Statistical analysis
The parameters of forensic interest were calculated using
the Powerstats program (http://www.promega.com). Estimation of the allele and haplotype frequencies, testing of
the Hardy–Weinberg equilibrium, analysis of molecularvariance, and calculation of RST values were performed
using the Arlequin program ver. 3.11 [5]. The linkage
disequilibrium coefficients D’ and r2 were calculated
between pairs of the STR and SNPs [6].

Results
STR typing
Figure 2 shows electropherograms of the allelic ladder used
for the STR typing and a sample with genotype 21–47.3.
The allelic sizes fell in a range from 130 to 300 bp. The
genotypes were clearly and unambiguously identified. A
total of 41 alleles were observed in 637 DNA samples
obtained from four populations and 54 families. These
alleles were classified into two groups, L and H, based on
size differences. Alleles 16–34, which belonged to group L,

were characterized by low molecular weights, whereas
alleles 40.3–55.3, which belonged to group H, werecharacterized by high molecular weights. A total of sevenmicrovariant alleles were found in group L. The Control
DNA 007 (Applied Biosystems) was identified as genotype
21–25.
Sequencing
To prepare the allelic ladder and to investigate the structureof the repeat motifs, a total of 113 alleles belonging to bothgroups were sequenced together with primate DNA(Electronic supplementary material Tables S1 and S2).
Common formulae for the repeat structures of groups L andH can be given as follows:group L :ðTTTCÞn CðTTTCÞ4

ðn ¼ 12  30Þ

group H : ðTTTCÞp ðTTCCÞq TTCðTTTCÞr CðTTTCÞ3ðp þ q þ r ¼ 37  52Þ
The marked differences between the two groups were dueto the insertion or deletion of TTCC units in an interme-

Fig. 2 Automated electropherogram of the CFI STR. (a) allelic ladder consisting of 31 bins for allelic products and 10 bins for mutants, and (b)genotype 21–47.3 observed in a JapaneseInt J Legal Med

diate region and of a TTTC unit in the 3′ region. The
microvariant alleles that contained a 0.3 repeat in group L
had the repeat structure of (TTTC)mTTC(TTTC)C(TTTC)4(m=21-27). In the microvariant alleles that contained a 0.2
or 0.1 repeat, the tetrameric TTTC repeat block wasinterrupted by a TT or T insertion, respectively. Somealleles had the same sizes, but differed in their nucleotide
and repeat structures. A total of 64 different sequences wereobserved in the 113 allelic products investigated. No hybrid
structure composed of the groups L and H sequences wasfound, suggesting that the increases and decreases in repeat
number have occurred by replication slippage but not byrecombination. The PCR products of the nonhumanprimates were smaller than those of humans. Amplificationof DNA from a Japanese monkey failed with the primers
CFI-i7F and CFI-i7R3 due to a deletion of several
nucleotides corresponding to the 3’ region of the primer
CFI-i7R3. PCR products were barely observed using theprimers CFI-i7F5 and CFI-i7R2. The sequences in the
primates were (TTTC)3(T/CTTC)T13-14.

Linkage analysis
Our family study has suggested that associations exist
between the group H alleles and c.1217A and between thegroup L alleles which contain a high number of repeats andc.603C. The genotyping results indicated that all of the
DNA samples which contained c.1217A also possessed atleast one of the group H alleles except for a Thai sample.
An association study was carried out between the group H
alleles and c.1217A. The group H alleles were combined
because the frequency of each allele was low. Table 1
shows haplotype frequencies and linkage disequilibriumcoefficients. Significant associations were found in the Thaiand Japanese populations. Similarly, c.603C was suggested
to be linked to the group L alleles with a high number of
repeats (alleles 29–34) and c.898A was suggested to belinked to the group L alleles with an intermediate numberof repeats (alleles 23–29).

Discussion
Family studies
Family studies including 54 parents and 57 offsprings were
performed and alleles were found to be inherited in a
regular Mendelian fashion. The results showed thatc.1217A (CFI*Ah) was inherited from parent to child
together with a group H allele. Similarly, c.603C (CFI*As)
was associated with alleles 30, 32, and 34.
Population study
Table S3 of the Electronic supplementary material shows
the allele frequencies and parameters of forensic interest for
the STR as well as the frequencies of the SNPs c.603C andc.1217A. The frequencies in the German and Japanese
populations were estimated using data from population
samples and parents. The CFI STR was highly polymorphic
in all populations investigated in this study. The Asian
populations had more alleles than the African and European
populations. The expected heterozygosity ranged from 0.89
in the German to 0.93 in the Thai populations. Interestingly,
group H was confined to Asian populations. Although eachof the group H alleles was low in frequency, a relatively
large number of alleles were observed. The sum of thefrequencies of the group H alleles was 0.162 in the Thaiand 0.116 in the Japanese populations, and these values
were slightly higher than those for c.1217A, which ischaracteristic of Asian populations [3]. Microvariant alleles
containing a 0.3 repeat were observed only in Africans andmicrovariant alleles containing a 0.2 repeat were only
observed in Japanese. Significant RST values were obtained
between the Asians and the other two populations.

Recent studies have suggested that all modern humans
originated from a single location in Africa where populations show the highest genetic diversity. A smooth loss ofgenetic diversity has occurred through bottleneck eventswith increasing distance from Africa. Genetic drift isgreater in Asians than in Europeans [7, 8]. The CFI STR
investigated in this study has a higher diversity in Asians
than in Africans and Europeans which comes from the factthat the Asian populations have population-specific alleles
that are classified into group H. A comparison of nucleotide
sequences between humans and nonhuman primates suggests that an increase in the tetrameric TTTC motif anddisappearance of polyT occurred along evolution of thehuman lineage. The divergence of Asians from Africans andEuropeans predated the acquisition of an ancestral allele of
the group H alleles in Asians. Subsequently, a c.G1217A
Table 1 Frequencies of haplotypes constructed with the c.G1217A
and groups H/L alleles and linkage disequilibrium values
Haplotype

Thai

Japanese

A-H
A-L
G-H
G-L
D′
R2
P

0.136
0.005
0.026
0.833
0.962
0.779
<0.001

0.104
–
0.011
0.884
1.000
0.890
<0.001

Group L alleles consist of alleles 16–34, and group H alleles, alleles
40.3–55.3

Int J Legal Med

mutation occurred in one of the group H alleles, and then
expanded into Asians. The c.A603C mutation must havearised on one of the group L alleles with a high number of
repeats.
The group H alleles are confined to Asian populations.
Although each of them is low in frequency, the sum of theirfrequencies was fairly high in the two Asian populations
investigated. They provide us with information on theancestry and population to which an individual belongs.
This study has also shown that the group H alleles areassociated with c.1217A (CFI*Ah). The frequency ofc.1217A is highest in the Han Chinese from Changsha,
Southern China and shows a south–north downward
geographical cline [3]. For further characterization of thegroup H alleles, therefore, investigation of their distribution
in Chinese and neighboring populations is important. The
association of c.1217A with the group H alleles suggeststhat an STR locus near a population-specific SNP may have
population-specific alleles.
The fibrinogen alpha chain (FGA) locus is located on
chromosome 4q28, about 45 Mbp distant from the CFI
gene. The FGA locus is hypervariable with about 80
alleles, which are also classified into low (alleles 12.1–35.2) and high (alleles 42.2–51.2) molecular weightgroups. However, the basic repeat structure is different
between alleles ≤30 and≥30.2. Some alleles are Africanspecific, but their frequencies are low [9, 10]. Unlike the
FGA locus, groups L and H in the CFI STR are clearly
distinguishable by their size and repeat structure.
The gene diversity of the CFI STR ranged from 0.89
in Germans to 0.93 in Thais. This STR shows highdiversity not only in Asians but also in Africans andEuropeans. These values are comparable to those of
hypervariable loci including FGA, D2S1338, and
ACTBP2 [11, 12]. The size of allelic products ranged
from 130 to 300 bp. It is easy to identify microvariants
with differences of 1 bp. In conclusion, the CFI STR is
unique in its hypervariability and possession of
population-specific alleles. The CFI STR would be auseful marker in forensic and anthropological sciences likeother autosomal STR markers which are not included incommercially available kits [13, 14].

Acknowledgements This study was supported in part by a Grant-inAid for Scientific Research (20590678 to IY) from the Japan Society
for the Promotion of Science.

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