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Introduction
The first component of complement Cl is
com-posed of three glycoprotein subcomponents, Clq, Clr and Cls, held together in a calcium-dependent complexi}. Clr js composed of a sjngle polypeptjde chain with a mol. wt. of 83,OOO and having 9.4%
carbohydrate by weight2). Genetic polymorphism of CIR was described by Kamboh and Ferrell in U.S. white and U.S. black blood donors, using
isoelectric focusing with immunoblotting3). It has
been suggested that observed phenotypes of CIR
are produced by a single autosomal locus with two
alleles, CIR"1 and CIR"2. Several new alleles,
CIR'3, CIR"4, CIR*5 and CIR*6, were
inves-tigated by Nakamura et al in the Japanese popula-tion, using thin-layer polyacrylamide gei
isoelec-tric focusing (PAGIEF) with enzyme
immuno-assay4). Other new rare alleles have also beenreported in various populations5)6}.
GENETIC POLYMORPHISMS OF HUMAN CIR SUBCOMPONENT
OF THE FIRST COMPLEMENT, FACTORIAND
FACTOR B IN CHINESE (LIAONING)
Xiuling WANG, Toshiko SAWAGUCHI and Shigeki NAKAMURA
Department of Legal Medicine (Director: Prof. Akiko SAWAGUCHI)
Tokyo Women's Medical College
(Received January, 12, 1994)Genetic polymorphisms of CIR subcomponent ef the first complement (CIR), factor I
(IF) and factor B (BF) subtypes were investigated in 360 healthy Chinese individuals
living in Liaoning by using polyacrylamide gel isoelectric focusing (PAGIEF) with
immunoblotting technique. In these samples, two new rare alleles were detected in CIR system, which were tentatively designated CIR*12 and CIR"13. In IF system a new rare allele was also detected and designated IF*M. The allele frequencies were estimated as follows. The CIR allele .frequencies were CIR*1=O.5181, CIR"2==O.3291, CIR*3==O.1472
and CIR*R (rare alleles)=O.O056, the IF al161e frequencies were IF"A=O.1055,
IF"B :O.8889 and IF*R=O.O056, the BF allele frequencies were BF"S==O.8208,
BF*F=O.1458, BF"FBI=O.O167, BF*S07 :O.O125 and BF"R=O.O042. The distribution of phenotypes fitted the Hardy-Weinberg equilibrium. The CIR, IF and BF systems providea useful genetic marker for human genetics, anthropological studies and forensic science in the Chinese population.
Factor I (C3b inactivator, IF) is an important
regulatory enzyme of the complement system,
mediating the proteolytic cleavage of the C3b and
C4b fragments, with complement component H,
C4-binding protein, membrane cofactor proteinand C3b/C4b receptor 1 as cofactors7). The genetic
polymorphism of IF was first demonstrated by
Nakamura and Abe using PAGIEF followed by
electroblotting with enzyme immunoassay8). They found twQ common alleles, IF"A and IF"B, in the Japanese population. Four new rare alleles, IF"C, IF*Al, IF"A2 and IF"Bl, have been identified in the Chinese and Japanese populations9)Ni3). The genetic polymorphism of factor B (BF) was demonstrated by Apler et al by using agarose gel electrophoresis followed by immunofixation with specific anti serum'`). Studies have indicated in large numbers of populations that the BF
pheno-types are determined by autosomal codominant
BF*F and BF*S, and two less common alleles,
BF"Fl and BF"Sl, the latter having been redesig-nated BF"S07 by Mauff et ali5}. Several rare alleles have also been reportedi5)ny2i}. Recently, subtypes of BF identified by means of isoelectric focusing have also been reported22)N26).
In the present investigation, the frequency
dis-tribution .of CIR, IF and BF alleles and new
variants detected in the Chinese population living in Liaoning are described.
Materials and Methods
1. Plasmasamples
Blood samples for population study were
ob-tained from 360 healthy Chinese living in
Liaon-ing. Plasma was collected by centrifugation at
3,OOO rpm for 10 min, and then 20 pt1 of
neura-minidase (40 units/ml, Fraction V, Sigma) was
added to 50 pt1 of plasma and incubated at 40C for
24 h. These samples were stored at -800C until
use.
2. Polyacrylamidegel(PAG)
Half millimeter thin-layer polyacrylamide gels
(T=:5%, C=3%) were prepared, containing 2.8%
ampholine carrier ampholate as described by
Nakamura et a14)i3)22}.
3. Isoelectric focusing electrophoresis
(IEF)IEF was carried out as follows with the LKB Multiphor II system (LKB, Sweden). Ten micro-liters of neuraminidase treated plasma samples was applied to the gel surface with Whatman 3
MM filter paper at a distance of 1.5 cm from the anodal end of the gel. For the phenotyping of BF, 5 pt1 of plasma samples was applied at a distance of 2.0 cm from the cathodal end of the gel. One mole
H3P04 (anode) and 1.0 M NaOH (cathode) were
used for the electrode solution. PAGIEF was
carried out at a constant power of 10 W,maxi-mum voltage of 1,OOO V, and unlimited mA, for 4 h at 40C, including prefocusing without samples for 40 min.
4. Immunoblottingprocedures
After PAGIEF was completed, immunoblotting
was carried out as follows. Before proceeding, a
nitrocellulose membrane (O.45 ptm, S&S, F.R.G)
was soaked for 1 h in a buffer consisting of 25 mM
Tris/192 mM glycine, 20% methanol, at pH 8.3. The proteins were transferred passively onto a
nitrocellulose membrane by leaving the
gel-membrane contact for 30 min under a weight of 1 kg.
5. Immunological detection of CIR, IF
and BF on the nitrocellulose membrane
After immunoblotting, the immune complexes
formed on the nitrocellulose membrane were
de-tected by using the method of Nakamura and Abe8) with slight modifications. For the phenotyping of
CIR, IF and BF, the goat anti human CIR serum
(Atlantic Antibodies, USA), goat anti human C3b
inactivator serum (Miles Labs, USA), goat anti human BF serum (Atlantic Antibodies, USA), and anti goat immunoglobulin-conjugated peroxidase
(DAKO Immunoglobulins Ltd., Denmark) were
used. The specific anti sera diluted 300 times with
5% bovine serum albumin phosphate buffered
saline (BSA-PBS) at pH 7.2 were mounted on the nitrocellulose membrane, and incubated at 370Cfor 1 h in a moisture chamber. The membrane was
washed with rocking for 5 min in O.05% Tween
20-PBS. Then the anti goat
immunoglobulin-conjugated peroxidase, which was diluted 600
times with 5% BSA-PBS, was mounted on the
membrane and incubated at 370C for 1 h in a
moisture chamber and washed as described above. Finally, nitrocellulose membrane was soaked in a solution consisting of 100 ml O.05 M Tris-HCI (pH
7.2), 25 mg 3-3'-diaminobenzidine
tetrahydro-chloride (Sigma, USA), and 10 ul 30% H202 for the
diaminobenzidine reaction. After CIR IF and BF
'
band patterns were detected, the reaction was
arrested by running tap water over the membrane for 5 min.
Results
1. CIRpolymorphism
Neuraminidase treated 360 plasma samples
were analyzed by PAGIEF with immunoblotting
technique. The band patterns of CIR phenotypesare shown in Fig. 1. Eight known phenotypes were
-411-observed: CIR 1-1, CIR 2-1, CIR 2-2, CIR 3-1, CIR 3-2, CIR 3-3, CIR 5-2 and CIR 6-2. This nomen-clature corresponds to that used by Nakamura et a14). Two new rare phenotypes were also detected in the Chinese population, which were considered to be controlled by two common and two new rare alleles. These new alleles were tentatively desig-nated CIR"12 and CIR*13. These new rare pheno-types were identical to CIR 12-2 and CIR 13-3, respectively.
The results of the population study are shown in Table 1. The allele frequencies were estimated to be O.5181, O.3291, O.1472 and O.O056 for CIR*1,
CIR*2, CIR*3 and CIR"R (CIR"5, CIR"6, CIR*12
and CIR*13), respectively. The observed and ex-pected values provided a good fit to the
Hardy-Weinberg equilibrium (x2=1.2074, df=3,
O.70<p<O.80).2. IFpolymorphism
In neuraminidase treated plasma samples from 360 Chinese subjects, PAGIEF band patterns were
classified into three common and three rare
phenotypes, and these band patterns were pre-sented in Fig. 2. These phenotypes werecon-sidered to be controlled by two common and three rare alleles. The two common alleles were iden-tical to IF*A and IF*B reported by Nakamura and Abe8}. The two rare alleles were also identical to
IF*Al reported by Nakamura and SawaguchiiO}
and IF*A2 reported by Dign et alii) and Nakamura et ali2). A new variant with a major band between IF A and IF B was designated IF M, and this new
Fig. 1 Band patterns of CIR phenotypes detected in 360 Chinese subjects by using polyacrylamide gel isolelectric focusing of neuraminidase treated plasma samples followed by immunoblotting with enzyme
lmmunoassay
Anode is at the top. Phenotypes from left to right: 2-2, 1-1, 5-1, 4-2 (reference sample), 1-1, 12-2, 2-1, 1-1, 3-2, 3-2, 6-2, 2-1, 3-3, 3-1, 5-2, 13-3, and 4-1 (reference sample).
Table 1 Distribution of phenotypes Chinese population
and allele frequencies of CIR in the
Phenotypes Observedno. o/o Expectedno. X2 Allelefrequencies
1-1 101 28,05 96,63 O.1976 CIR.1=O.5181 2-1 120 33,33 122.76 O.0621 CIR*2=O,3291 2-2 40 11.11 39.00 O.0256 CIR*3=O.1472 3-1 51 14,17 54,91 O.2784 CIR*R=O.O056 3-2 34 9,44 34.90 O.0232 3-3 10 2,78 7.80 O.6205 O,70<p<O.80 5-2 1 O,28 (df=i3) 6-2 1 O,28 12-2 1 O,28 4.00 o,oooo 13-3 1 O.28 Others o o.oo Total 360 100.00 360.00 1,2074
Fig. 2 Isoelectric focusing patterns of IF phenotypes detected in 360 Chinese subjects
Anode is at the top. Phenotypes from left to right: AIB, B, B, A, A2B, A, AB, A, A2B, AB, and BM.
Table 2 Distribution of Chinese population
phenotypes and allele frequencies of IF in the
Phenotypes Observedno. o/o Expectedno. X2 Allelefrequencies
AABBAIBA2BBMOthers 1,39 18.33 79.16 O,28 O.56 O,28 o.oo 4.01 67.52 284.45 4.02 O.2444 O.0342 O.OOIO O.OOOI IF*A=O.1055 IF*B=O,8889 IF*R=O.O056 O.80<p<O.90 (df=2)
Total 360 100,OO 360.00 O.2797
IF'R:the combined of IF'Al IF'A2 and
'
IF.Mallele was also designated IF*M.
Table 2 presents the distribution of phenotypes and allele frequencies of IF in 360 Chinese indi-viduals. The allele frequencies were estimated as
IF*B=O.8889, IF"A=O.1055 and IF"R (IF"Al,
IF*A2 and IF"M)=O.O056, respectively. Observed phenotype numbers did not differ significantly from those expected, assuming a Hardy-Weinberg equilibrium ix2=O.2797, df=2, O.80<p<O.09).
3. BFpolymorphism
Figure 3 shows the band patterns of BF pheno-types detected in 360 Chinese subjects by using
PAGIEF of plasma samples followed by
immuno-blotting with enzyme immunoassay. PAGIEF
band patterns were classified into three commonphenotypes, BF SS, BF FS and BF FF, and five variant phenotypes, BF S07, BF SS07, BF FS07,
BF F075S and BF F025S. The subtype of BF F was also detected and found to be identical to BF FBIS. The distribution of phenotypes and allele fre-quencies are shown in Table 3. Allele frefre-quencies
calculated for these 360 individuals were O.8208, O.1458, O.O167, O.O125 and O.O042 for BF"S, BF"F,
BF"FBI BF"S07 and BF*R, respectively. The
distribution of phenotypes fitted the Hardy--Wein-berg equilibrium ix2=2.3999, df=4, O.50<p<O.70).
Discussion
The genetic polymorphism of human CIR had
first been described in U.S. whites and U.S.blacks3}, and it has been suggested that observed
phenotypes are produced by a single autosomal
locus with two alleles, CIR"1 and CIR*2. An
additional four alleles, CIR"3, CIR*4, CIR*5 and CIR"6, have also been reported in the Japanese population4). CIR"1 is the most common allele in
Caucasoids and Negroids whereas in Mongoloids
CIR"1 varies between O.42 and O.52. In the pre-sent study, we found the common alleles, CIR"1 and CIR*2, and variant alleles, CIR*3, CIR*5 and CIR"6, but CIR*4 was not detected. Furthermore,
Fig. 3 Isoelectric focusing patterns of BF phenotypes detected in 360 Chinese subiects
Anode is at the top. Phenotypes from left to right: FS07, F075S, SS07, FBIS, S07, SS07, FS, FF, SS, SS, FS, FS, SS, FS, FBIS, SS, FF, FS, SS, SS, and F025.
FBIS,
Table 3 Distribution of Chinese population
phenotypes and allele frequencies of BF in the
Phenotypes Observedno. o/o Expectedno, X2 Allelefrequencies
ss 244 67.78 242.54 O.O088 BF*S=O.8208
FS 84 23.33 86.16 O,0542 BF*F=e.1458
FF 10 2.78 7.65 O.7219 BF*FBI=:O,O167
SS07 6 1,66 7.39 O.2614 BF*S07=O.O125
FBIS 12 3.33 9,87 O,4597 BF*R=O.O042
FS07 1 O.28 O,50<p<O,70 S07 1 O,28 (df=4) F075S 1 O.28 6.39 O.8939 F025 1 O.28 Others o o.oo Total 360 100.00 360,OO 2,3999
BF'R : the combined of BF'F025 and BF'F075
observed in the Chinese population. A comparison of CIR allele frequencies in different populations is indicated in Table 4. The allele frequencies in the Chinese and Japanese populations are similar. It is interesting that the Mongoloids have a large
genetic variation in CIR compared with
Cauca-soids and Negroids.
IF allele frequencies in various populations are
summarized in Table 5. The IF"A allele which
occurs in Mongoloids has a very low frequency in
Table 4 Comparison of CIR allele frequencies in different populations
Allelefrequencies
Populations Refs,
CIR*1 CIR*2 CIR*3 CIR*4 CIR*R
U.S.Whites O.934 O.066 3
U.S.Blacks O,899 O,101 3
Nigerianblacks O,816 O.164 O.O16 O.O04 5
MayanIndians O.945 O.029 e.o26 5
DogribIndians O.832 O,168 5
Aleuts O.893 O,067 O.040 5
St.LawrenceIslandInuit O.708 0.139 O,153 5
KodiakIslandInuit O.887 O.082 O.027 O.O04 5
JapaneseTokyo O.422 O.360 O.207 O.O09 O.O02 4
JapaneseYamanashi O.456 O,338 0.195 O.O09 O.O02 6
Table5 Comparison of IF allele frequencies in different populations
Allele frequencies
Populations Refs.
IF*B IF*A IF*R French O.994 O,O06 27
Nepalese O,942 O.058 27
Korean
ChejuIsland O.908 O.092 11
Japanese
Iwate O.896 O,102 O,O02 12
Yamagata O.899 O.098 O,O03 11
Tokyo O.892 O.107 O,OOI 10
Yamaguchi O.880 O.120 27
Oita O.874 O,126 28
Chinese
Taiwan O.929 O.071 27
Shenyang O.901 O,099 11
Liaoning O.889 O,106 O.O04 Presentstudy
Caucasoids. It has been indicated that a west-to-east geographical cline for allele frequencies of IF exists in Eurasia, ranging from France to western Japan27). Furthermore, the existence of a statis-tically significant geographical cline for allele frequencies of IF*A and IF*B in the Japanese main islands has also been indicatedi2}. The highest frequency for IF*A has been observed in western Japan. In the present study, two common alleles,
IF*A and IF*B were observed, and allele
fre-quencies were estimated as IF"A=O.1055 and
IF*B =O.8889. These frequencies were essentially
the same as those indicated in the Japanese
population. So it was confirmed that ageogra-phical cline for allele frequencies of IF exists in Eurasia. Two rare alleles, IF"Al and IF"A2, have
only been observed in the Chinese and Japanese populations, and new rare allele IF"M has also been detected in the Chinese population. These
results suggested that the IF system shows a
higher genetic variation in Mongoloids than inCaucasoids. So we can assume that the IF system is a useful genetic marker for human genetics and anthropological studies.
The frequencies of common and rare BF alleles
in the major races are included in Table 6. BF"S is
the Predominant allele in Caucasoids and
Mon-goloids whereas in Negroids BF"S varies between O.28 and O.44. Variant forms with faster or slower
mobilities are rare in all populations. In the
present study, we have detected two common
alleles, BF"S and BF"F, and three rare alleles,
BF*S07, BF*F075 and BF"F025. BF"S07 was
detected in Caucasoids, but not in the Japanese population. It was anthropologically interesting that BF"S07 allele, which is very rare iri Mon-goloids, exists in the Chinese population, with theallele frequency of BF"S07 being a polymorphic
frequency.
Teng and Tan23) and Geserick et a124} first
reported on the detection of BF F subtypes inisoelectric focusing. They were found to be
com-mon, but shared the major F band. Weidinger et
al25> also reproted on BF S subtypes, BF Sbl and
BF Sb2 being rare. Subsequently, Nakamura et
a122) demonstrated a new BF F subtype which
Table 6 Comparison of BF allele frequencies in different populations
Allelefrequencies
Populations Refs.
BF*S BF*F BF*S07 BF*FBI
BF.R
U,S.Whites O,709 O.278 O.OI3 14
German O,808 O.174 0.009 O.O09 29
U,S.Blacks O.437 O.512 O,051 14
NegroidSouthAfrican O.282 O,655 O.063 30
Filipinos O.703 O.297 31
Thailanders O.847 O.153 31
Indians O.698 O.290 O.O12 31
JapaneseTokyo O.795 O.182 O,022 O.OOI 22
Osaka O.834 O.149 O,O15 O.O02 26
ChineseLiaoning O.821 O.146 O.O12 O,O17 O.O04 Presentstudy
-415-appears unlike all previous ones. By using iso-electric focusing, a BF F subtypes was observed in the Chinese subiects, and this allele was identical to BF"FBI reported by Nakamura et a122). It has been assumed that BF*FBI js a Mongoloid-specific allele just as is the second component of
comple-ment C2*AT.
For the forensic application of CIR, IF and BF systems, the distinguishing probabilities (D.P.) were calculated as O.7681, O.3396 and O.4841 for CIR, IF and BF, respectively. These results
sug-gested that CIR, IF and BF systems are useful
genetic markers for medicolegal identification and .
paternity tests as well as anthropological studies.
Acknowledgment
I wish to express my sincere gratitude to Professor Dr. Akiko Sawaguchi for providing me with necessary facilities and constant encouragement during the tenure
of my research work. I would also like to thank the rest
of the Department of Legal Medicine for providing a friendly and stimulating atmosphere to work in.
This work was supported by Uehara Memorial
Foundation.
A part of the present paper was presented at the 62nd Kanto District Medico-Legal Conference in Tokyo in l993.
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