The investigation of allele and genotype frequencies of humanC3 (rs2230199) in south Iranian population

Najmeh Bazyar • Negar Azarpira •

Saied Reza Khatami • Hamid Galehdari

Received: 2 December 2011 / Accepted: 7 June 2012 / Published online: 21 June 2012

� Springer Science+Business Media B.V. 2012

Abstract The complement system is an important

mediator of natural and acquired immunity. The comple-

ment system genes coding complement proteins have

polymorphisms. Hereditary deficiencies of this system

predispose to autoimmune conditions such as age-depen-

dent macular degeneration or impairment of immunity

against microorganisms. When different populations are

compared, the frequency of complement polymorphism

shows a very marked geographical distribution. The

frequency of the functional polymorphism rs2230199

(Arg80Gly; C [ G) in the C3 gene was determined in

population from south of Iran (n = 200), using polymerase

chain reaction-restriction fragment length polymorphism

(PCR–RFLP). One hundred thirty-eight persons (69 %)

were homozygous for C allele (CC or SS); fifty-six person

(28 %) heterozygote GC (FS) and six people were homo-

zygous for G allele (GG or FF) (3 %). The allele frequency

was 82 % for C3S and 18 % for C3F. A distribution of

C3C allele frequency in our population is different from the

reports of Asians (100 %); Indians (90–98 %); African-

American (93 %); Africans (99 %) and south Brazilian

(97 %). However, this finding is similar with the findings

Caucasian (80–82 %) (http://www.ncbi.nlm.nih.gov/SNP);

Americans (80 %); Pushtoon, Hazaras, Osbek and Tajik

ethnic groups in Afghanistan (88–90 %) and Tunisian

population (84 %). Our study confirmed significant inter-

ethnic differences in C3 (rs2230199) frequencies between

south Iranians and other ethnic groups. The analysis of

genetic variation in complement genes is a tool to provide

new insights into the evolution of the human immune

system.

Keywords C3 gene � Polymorphism � South Iranian

Introduction

The complement system is an important mediator of

natural and acquired immunity in human. It consists of

approximately 30 different proteins with catalytic activity,

function as regulators, or act as cellular surface receptors.

These components normally circulate in the serum in

inactive forms and are activated by three different path-

ways: classical, alternative or lectin. Complement compo-

nent 3 plays a central role in all three activation pathways

[1].

C3 is an acute phase reactant; increased synthesis of C3

is induced during acute inflammation. The liver is the main

location of synthesis, but small amounts are also produced

by activated monocytes/macrophages. Component C3

plays several important biologic roles in the activation

complement pathways such as formation of C3 and C5

convertases; production of opsonin for enhancement of

microorganism phagocytosis; clearance of C3b-bound

immune complexes and clearance of apoptotic cells [1].

Wieme et al. [2] found a double electrophoretic band

corresponding to complement component C3, by using

high voltage starch gel electrophoresis. Azen et al. [3] also

found electrophoretic polymorphism of the third compo-

nent of complement.

Human C3 gene is located on chromosome 19 and

exhibit genetic polymorphism. A common functional

N. Bazyar � S. R. Khatami � H. Galehdari

Department of Genetics, Faculty of Science,

Shahid Chamran University of Ahwaz, Ahvaz, Iran

N. Azarpira (&)

Transplant Research Center, Shiraz University

of Medical Sciences, Shiraz, Iran

e-mail: negarazarpira@yahoo.com

123

Mol Biol Rep (2012) 39:8919–8924

DOI 10.1007/s11033-012-1759-9

polymorphism rs2230199 (Arg80Gly; C [ G) in the C3

gene, is corresponding to the electrophoretic variants C3S

(slow) and C3F (fast) [4]. C3S allele is the most common

allele in all populations.

Botto et al. [5] studied the molecular basis of the C3F

versus C3S polymorphism. The less common variant, C3F,

occurs with appreciable frequencies (gene fre-

quency = 0.20) only in the Caucasoid populations. They

found a single nucleotide change, C-to-G, at position 364

in exon 3, distinguishing C3S and C3F [5]. This led to a

substitution of an arginine residue in C3S for a glycine

residue in C3F (R102G). The substitution resulted in a

polymorphic restriction site for the enzyme HhaI. The risk

allele G seems to be geographically confined to Europe

(0.17) is rare in Africa and absent in Asia. However,

available genetic and functional data does not explain the

geographical distribution of this C3 polymorphism [4, 5].

This SNP has many important functions in immune

mechanisms. The presence of F allele (C3F) has been

shown to be associated with different immune diseases

such as IgA nephropathy [7], Systemic Vasculitis [8].

Recent studies have suggested that C3 alleles can influence

the outcome of kidney transplantation [9].

Yates et al. [10] genotyped SNPs of the complement

genes C3 and C5 in Caucasian English patients with age-

related macular degeneration and found that the common

functional polymorphism in the C3 gene (rs2230199) was

strongly associated with age-related macular degeneration

(ARMD). The association was replicated by other groups

[11, 12].

Since this polymorphism is associated with few disease,

such as ARMD, and no report from our country is available

in the literature, we analyzed 200 healthy individuals to

determine rs2230199 variants and identify the frequency of

alleles in south Iranian population.

Materials and methods

To examine the allele frequencies of C3 gene (rs2230199),

200 unrelated individuals, aged from 35 to 60, from Shiraz

(i.e., a city located in Fars province, south of Iran) were

selected randomly from Shiraz Blood Transfusion Organi-

zation. The subjects who had renal or liver disease or any

other chronic illnesses were excluded from the study. As this

SNP is associated with ARMD, any participant with this

degenerative disease was excluded from study. In addition,

prior to the commencement of the research, Ethics Com-

mittee approval and participants’ consent were obtained.

DNA was extracted from buffy coat by using of com-

mercial DNA extraction kit (Gene fanavaran, Iran) and

genotyped by polymerase chain reaction-restriction frag-

ment length polymorphism (PCR-RFLP) method. Primers

were designed with the Fast-PCR program. The Forward

primer’s sequence was: 50CTCACCTGTGGAGCCAGGG

GTGTA-30 and the Reverse primer’s sequence was: 50-CC

AAAACGGCCACCTCGGAAGACC-30. The PCR was

carried out in 25 ll solution consisting of 40–50 ng DNA,

1.5 mM dNTP, 10 mM Tris HCL, 1.5 mM Mgcl2, 10 pmol

each primer and 1 U Taq polymerase.

PCR conditions were as follow: initial denaturation at

95 �C for 7 min followed by 35 cycles of 95 �C for 1 min,

64 �C for 1 min and 72 �C for 30 s to amplify a 300 bp

product. The HhaI was used as restriction endonuclease.

Samples with C3FF genotype produced a 300 bp prod-

uct, C3SS genotype yielded 167 and 133 bp fragments

after enzyme digestion and the heterozygote C3FS had 3

bands.

In addition, few samples from each genotype were

sequenced with a Genetic Analyzer (Applied Biosystems,

Foster City, CA) that confirmed the PCR-RFLP results.

Statistical analysis

First, the gene counting method was used to estimate allele

frequencies. Then, the data were analyzed by using Chi-

square and Fisher’s exact test. In addition, the observed

genotype frequencies were compared with the expected

genotype frequencies according to the Hardy–Weinberg

equilibrium with Arlequin software (version 3.1). Differ-

ences were considered significant at P \ 0.05. SPSS, ver-

sion 16.0, was used for the analysis of the statistical data.

Results

The distributions of the genotypes and allele frequencies

C3 gene (rs2230199) and comparison with other population

were summarized in Table 1. The C3C allele was found in

164 persons with allelic frequency 0.82 (heterozygote 55,

homozygote 137), which is the most common allele among

Caucasians (80–82 %) [6]. The frequency of 82 % is dif-

ferent (P \ 0.05) from the reports of Asians (100 %) [6];

Indians (90–98 %) [13, 14]; African-American (93 %) [6];

Africans (99 %) [6] and south Brazilian (97 %). However,

this finding is consistent (P [ 0.05) with the findings

Caucasian (80–82 %) [6]; Americans (80 %) [15]; Sardi-

nians (an island in the Mediterranean Sea) (77 %) [16];

Italians (80 %) [17]; Pushtoon, Hazaras, Osbek and Tajik

ethnic groups in Afghanistan (88–90 %) [18] (Fig. 1);

ESP-Cohort populations (86 %) [6], and finally Tunisian

population (84 %) [19].

These results suggest that allelic distribution in Fars

province, south of Iran, is more similar to European and

neighborhood countries than Asian, Indian or Africans.

8920 Mol Biol Rep (2012) 39:8919–8924

123

The genotype frequency in our population is in Hardy–

Weinberg equilibrium. PCR-RFLP results for the C3S/F

polymorphism are shown in Fig. 2.

Discussion

Genetic and archaeological evidence indicate that humans

emerged in Africa about 100,000–200,000 years ago and

colonised other continents 60,000 years ago [20], followed

by diversification to non-African populations 23,000 years

ago [21–23].

In order to generate different molecular ‘‘signatures’’ in

the patterns of genetic variation, three forms of natural

selection may be take place. Negative or purifying selec-

tion is when deleterious alleles are removed from a popu-

lation. These mutations are generally happen at low

frequencies and is responsible for the removal of mutations

associated with severe Mendelian disorders [24, 25].

Positive or directional selection refers to selection of

advantageous mutations, results to high frequency of

selected alleles within the population at a faster rate than

neutrally evolving allele. A third form of natural selection

is termed as balancing selection, in which heterozygotes

show a higher frequency than homozygotes and leads to the

maintenance of alleles in a population at a given locus [26].

Andres et al. believed that some genes associated with

immune function showing signatures of long-term balanced

selection [26, 27].

The effect of genetic variation in complement genes on

the development of disease has been reported. The fre-

quency of disease-associated polymorphisms of comple-

ment system is different in geographically disparate

populations. An example is the polymorphism Ile62Val

(rs800292 (A [ G)) in the complement Factor H gene

which associated with age-related macular degeneration

(ARMD), that the Ile62 polymorphism protects against

AMD [23, 28]. However, the frequency of this polymor-

phism shows a very marked geographical distribution

between sub-Saharan African and European populations.

Polymorphisms in other complement genes such as com-

plement factor B also has similar trends [23].

Mannose-binding lectin (MBL2 gene) is a soluble pro-

tein that binds microorganisms and activates the lectin

complement pathway [29]. Genetic analysis of the MBL2

gene has revealed the existence of three common poly-

morphisms in exon 1 resulting in three variant alleles

termed, B, C and D; the normal human variant is A. All

three alleles affect the level of MBL in serum with 90 %

decrease in MBL function [29].

Homozygous carriers of MBL2 variant alleles are at

substantially increased risk of pneumococcal disease. [30].

Table 1 Distribution of C3 gene (rs2230199) genotypes and alleles among different ethnic groups

Alleles (%) P value OR 95 % CI Genotype (%) P value Reference

C (slow,

S)

G (fast,

F)

CC

(SS)

CG

(FS)

GG(FF)

African-American 93 7 0.018* 2.92 1.08–8.15 86 14 0 0.008* [6]

European 82 18 1.00 1.06 0.46–2.18 70 25 5 0.71 [6]

Asian 100 0 \0.001* 21.73 2.96–4.46 100 0 0 \0.001* [6]

Sub Sahara African 99 1 \0.001* 21.73 2.96–4.46 98 2 0 \0.001* [6]

ESP_ Cohort Population 86 14 0.44 1.35 0.59–3.09 73 24 3 0.81 [6]

Kaingang Indian South

Brazil

98 2 0.0001* 10.76 2.3–69.21 – – – – [13]

South India (Kotas/Niilgiri) 90 10 0.10 1.98 0.81–4.91 – – – – [14]

USA Ohio 80 20 0.71 0.88 0.41–1.88 65 29 6 0.56 [15]

Sardinia 77 23 0.38 0.73 0.35–1.55 – – – – [16]

Italy 80 20 0.71 0.88 0.41–1.88 – – – – [17]

Afghanestan Tajik 85 15 0.56 1.24 0.55–2.81 – – – – [18]

Pushtoon 88 12 1.41 1.61 0.69–3.81 – – – –

Hazaras 90 10 0.10 1.98 0.81–4.91 – – – –

Osbek 85 15 0.56 1.24 0.55–2.81 – – – –

Tunisia 84 16 0.14 1.15 0.52–2.57 – – – – [19]

South of Iran 82 18 69 28 3 Current

Study

– The genotype frequency is not available in literature

* Significant differences

Mol Biol Rep (2012) 39:8919–8924 8921

123

Although this finding was not replicated in other studies

from Europe [31, 32]. MBL2 variants may also associate

with susceptibility to leprosy in Brazilian patients [33].

In contrast, no association was observed between these

polymorphisms and leprosy in an African cohort from

Malawi. The same controversial association has been

reported on MBL variants and susceptibility to tuberculosis

[33–35].

It seems that protective MBL2 variant within Asians

(protective against leprosy in the Nepalese population) is

maintained by balancing selection (advantage for hetero-

zygous individuals) as with the classical example of the

sickle cell trait (HbS allele) in sub-Saharan Africa [23].

Thakkinstian et al. did a systematic review and meta-

analysis on complement polymorphism and ARMD. The

minor allele (C3G) frequencies for rs2230199 range

from 3.2 to 6.8 % in African Americans, 16.9–20.6 % in

Europeans, and 0.8 % in sub-Saharan Africans. Subgroup

analysis by ethnicity indicates that C3 variant was very

infrequent in Asians.

They concluded that for rs2230199, patients with CG

and GG genotypes were 1.44 (95 % confidence interval

Fig. 2 PCR-RFLP results for the C3S/F polymorphism

Fig. 1 Iran and Afghanistan are two neighborhood countries with common ancient ethnic

8922 Mol Biol Rep (2012) 39:8919–8924

123

(CI) 1.33, 1.56) and 1.88 (95 % CI 1.59, 2.23) times more

likely to have ARMD than patients with the CC genotype

[28].

Another study evaluated the association between com-

plement component 3 (C3F) and risk of postoperative

neurocognitive dysfunction following carotid endarterec-

tomy. The complement cascade plays a central role in

ischaemia–reperfusion injury following surgery.

They believed that C3F polymorphisms are strong

independent predictors of moderate-to-severe neurocogni-

tive dysfunction at 1 day following carotid endarterectomy

[36].

The findings of this study, regarding of common func-

tional polymorphism rs2230199 (Arg80Gly; C [ G) in the

C3 gene indicate significant similarities and differences

between the frequency distribution of major allelic varia-

tions among different ethnics which affects the suscepti-

bility of humans to different disease i.e. infectious disease

and/or ARMD. Our frequency is resemble Caucasians,

Americans and different from Asians, Africans and

Indians. This frequency is also similar to report from

Afghanistan, a neighborhood country. Afghanistan and Iran

are historically tied to create Persian civilization. After

2000 BCE, people from Central Asia moved south into the

boundaries of modern Afghanistan, among them were

many Indo-European-speaking Indo-Iranians [37]. These

people later migrated west to what is now Iran, and towards

Europe via the area north of the Caspian. This region was

called Ariana [37].

Iran is located in the Middle East and between Oriental

and European populations, it has a highly admixed popu-

lation due to invasion and/or migration [38–40]. On the

other hand, Afghanistan is also between Central Asia, the

Middle East and the Indian sub-continent with a nation

made up of many different nationalities. Therefore it is

logical to find similar genetic backgrounds between these

two ancient countries.

However in order to study patterns of variation within

complement genes and search for signs of selection more

genome-wide and denser SNPs datasets is needed.

Conclusion

The C3 (rs2230199) allele frequency in our population is

resemble Caucasians, Americans and different from

Asians, Africans and Indians and also similar to report

from Afghanistan, a neighborhood country. Its distribution

is different from Asians, Indians and Africans. Our

study confirmed significant inter-ethnic differences in C3

(rs2230199) frequencies between south Iranians and other

ethnic groups.

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