Journal of Life Sciences Volume 5 Number (3)

J LS

Journal of Life Sciences

Volume 5, Number 9, September 2011 (Serial Number 41)

Contents

Research Papers

Statistical Analysis of Genetic Diversity in 15 STR Loci from Han, Miao and Yao Tribes in South China

Kuanheng Wu, Miao He, Yijing Wang, Jian Song, Liping Ling and Daniel Wai Tin Chan 682

CCR5-CCR2 Gene Polymorphisms in Ethiopian Jews: Population Divergence and Its Relevance to HIV-1 Infection Resistance

Michael Korostishevsky, Batsheva Bonne-Tamir, Zvi Bentwich, Alexander Kalinkovich and Alexander Tsimanis

690 First Record of Frankliniella Occidentalis and Impatiens Necrotic Spot Virus in Egypt

Abeer Salah El-Deen Abd El-Wahab, Mohamed Abdel-Kader El-Sheikh and Salah Elnagar

697 Investigation of the Optimum Condition and Antimicrobial Activities of Pigments from Four

Potent Pigment-Producing Fungal Species

Neveen S. Geweely

712 Effect of Ginger Powder (Zingiber Officinale) on Plasma Lipid Profile and Liver Enzyme Activities of Hypercholesterolemic Rats

Ajayi Olubunmi Bolanle 717

Parasitic Plants as a New Target Plant for Screening Rice Allelopathic Potential

Yiqing Guo, Kil-Ung Kim, John I. Yoder and Donghyun Shin 725

Effect of Graded Levels of Nitrogen on Growth and Yield of Eggplant (Solanum Melongena) in Kabba, Southern Guinea Savanna Ecological Zone of Nigeria

John Akintola Oloniruha

728 Alternative Technologies Used in Laying Hens Husbandry

Marius Giorgi Usturoi, Paul Corneliu Boi şteanu, Răzvan Mihail Radu-Rusu, Ioan Mircea Pop, Marius Gheorghe Doli ş and Alexandru Usturoi

733

Phylogenetic Study of Freshwater Bivalve Parreysia Corrugata from Maharashtra State, India by 18S rRNA Sequences

Madhav V. Upadhye, Rajesh C. Patil, Sonal M. Manohar and Ujwala Jadhav 739

Effect of Thermal Stress, Cistern Size and Milking Frequency on Plasma Mineral Concentrations in Holstein Dairy Cows

Rim Ben Younes, Moez Ayadi, Taha Najar, Margherita Caccamo, Iris Schadt and Moncef Ben M’Rad 747

Effect of Ageing Time on Meat Characteristics of Castrated and Uncastrated Brahman Cattle

José A. Miguel, Jesús Ciria, Begoña Asenjo, David Colmenarez and Hector Pargas 754

Effect of Climate Change on Phenology of Forage Grass Species

Żurek Grzegorz

759 Developing Adaptation Strategies Due to Climate Change: With Special Reference to the Vulnerable Java Fisheries, Indonesia

Indah Susilowati and Agus Hartoko 768

Fast Determination of Cd, Pb, and Cu in Grape Must and Wine

Jarmila Lastincova, Ernest Beinrohr and Lubica Pospísilova 772

Effect of the Chromatic Assimilation (Bezold Effect) in the Vision of the Content on a Dinner Plate

Ignacio Tortajada, Jorge Montalvá and Mariano Aguilar

Journal of Life Sciences 5 (2011) 677-681

Statistical Analysis of Genetic Diversity in 15 STR Loci from Han, Miao and Yao Tribes in South China

1 1 1 1 2 Kuanheng Wu 3 , Miao He , Yijing Wang , Jian Song , Liping Ling and Daniel Wai Tin Chan 1. Life Sciences School, Sun Yat-sen University, Guangzhou, Guangdong 510275, China

2. Education School, Sun Yat-sen University, Guangzhou, Guangdong 510275, China 3. Department of Building Services Engineering, The Hong Kong Polytechnic University, Hunghom, Hong Kong, China

Received: August 18, 2010 / Accepted: November 03, 2010 / Published: September 30, 2011.

Abstract: It is interesting to find the possible statistical characters of 15 STRs in Han, Miao and Yao, the three main tribal populations in South China, and the significant differences of allele frequencies by comparing STR loci in each cluster for applications of forensic science. Genetic diversity in 15 STR loci [D8S1179, D21S11, D7S820, CSF1PO, D3S1358, TH01, D13S317, D16S539, D2S1338, D19S433, vWA, TPOX, D18S51, D5S818, and FGA] from Han, Miao and Yao tribes in South China had been analyzed. The allele frequencies of 15 tetrameric STR loci were obtained from 1,530 unrelated individuals of three main tribal populations [Han, Yao and Miao] inhibiting in South China. Cluster analysis and LSD test had been used for data analysis. The high degree statistical differentia of genetic polymorphism has been found among three tribal populations.

Key words: STR, genetic diversity, statistics, Han, Miao, Yao.

Abbreviations: STR: short tandem repeats; The traditional STR loci applied in forensic science includes 15 STR loci [D8S1179, D21S11, D7S820, CSF1PO, D3S1358, TH01, D13S317, D16S539, D2S1338, D19S433, vWA, TPOX, D18S51, D5S818, and FGA]; LSD: a least significant difference test of one-way analysis of variance.

1. Introduction differences founded at specific STR loci among Chinese populations in different locations. For

15 STR loci from three tribal populations Han, example, the significant differences were found at Miao and Yao in South China were obtained in present three STR loci [D3S1358, D21S11, D8S1179] study. Whole DNA extracted from venous blood was between Sichuan and Beijing populations, [D3S1358, obtained from randomly unrelated 1,000 Han, 248 D21S11, TPOX] between Sichuan and East China Yao and 282 Miao individuals, Guangdong Province. populations, [D5S818, D13S317, CSF1PO] between Tribe Miao and Yao have over nine million people Sichuan and Hong Kong populations according to the inhabiting mainly in mountainous areas of Guizhou, study of Zhang et al. [1]. It is interesting to find the Guangxi and Hainan Province. possible statistical characters of 15 STRs in Han, Miao The traditional methods of Hardy-Weinberg and Yao, the three main tribal populations in South equilibrium, the power of discrimination, the China, and the significant differences of allele probability of paternity exclusion, and the frequencies by comparing STR loci in each cluster for polymorphic information content have already applications of forensic science. Statistical methods presented profound conclusions about the significant based on cluster analysis, as well as LSD test show the

significant differentiation among the three tribal Corresponding author: Miao He, Ph.D., associate professor, research fields: bioinformatics, biostatistics. E-mail: populations. The aims of this study are to approach: (1)

lsshem@mail.sysu.edu.cn.

Statistical Analysis of Genetic Diversity in 15 STR Loci from

Han, Miao and Yao Tribes in South China

the genetic polymorphism presented by cluster analysis tribes. Further variance analysis was applied on these especially the genetic diversity between Han and two

STR loci using LSD value, calculated from two sets of other tribes; (2) a region of allele frequencies selected

STR locus allele frequencies array or mean allele by LSD test revealing significant differences among

frequencies array of each allele from several STR loci STR loci in the same cluster could serve as a genetic

in the same cluster, to discriminate the significant marker for individual identification among three tribal

differences of allele frequencies between two STR loci populations; (3) collecting the genetic polymorphic

[12, 13]. To be specific, we calculated LSD (= 0.05) information from the three tribal populations and

value from two arrays {X 1 ,X 2 ,X 3 …} and {Y 1 ,Y 2 , retrospectively interpreting the demographic tribal

Y 3 …} which represented allele frequencies of STR migration and amalgamation in South China.

locus X and Y separately. If |X i -Y i | > LSD, the allele frequencies Y i is significantly different from X i .

2. Materials and Methods

Microsoft Office Excel 2007 and SPSS 15 were used to

2.1 DNA Analyzed

analyze these data.

The allele distributions of 15 STR loci were obtained

3. Result

from 1,530 unrelated individuals [2-6]. Genome DNA Cluster analysis preformed on correlation matrix was extracted from whole blood using Chelex-100 indicated several profound conclusions which were extraction method (Bio-lad Company). Those samples then further studied by using LSD test. The results of

failed in former method using standard phenol

hierarchical cluster analysis revealed highly chloroform methods or DNA IQ commercial kits

TM

(Promega Company) extract DNA again [7]. consistent clusters iv among three tribal populations and clusters [i, ii, iii] shared genetic diversity to

2.2 STR Typing variant extend. LSD test applied on three STR loci

All samples were PCR amplified using [D8S1179, D7S820, D13S317] of Han further AmpFlSTR® Identifiler™ commercial kits (Applied

corroborated the significant diversity of clusters [i, Biosystems Company) [8]. PCR amplifications were

iii] among Han, Miao and Yao.

carried out according to manufacturers (AmpF/STR

3.1 Cluster Analysis

Indetifi-ler PCR amplification kit, AB Applied Biosystems). Amplification products were run in an

The hierarchical cluster analysis based on ABIPRISM 3100 genetic analyzer (ABI Company)

correlation matrix revealed profound genetic diversity using Liz500 as internal standard label. Genescan 3.7

characteristic among three tribes. 15 STR loci from (ABI Company) software was used to collect the data,

each tribe were subdivided into two primary clusters [I, analyze fragment sizes and Genepop v3.4 to calculate

II] and four secondary clusters [i, ii, iii, iv] separately the allele frequencies of each STR locus in 3 tribes [9].

in Fig. 1. The members in each cluster [i, ii, iii, iv] were nearly exactly the same, except three locus

2.3 Statistical Analysis [D8S1179, D7S820, D13S317] drift or switch,

Cluster analysis was applied on 15 STR loci from however the topologic structures differed obviously each tribal population using the correlation matrix

among three tribes especially between clusters iii of Han calculated from 15 sets of STR loci allele frequencies

and Yao, Han and Miao. These dissimilarities found [10, 11]. By comparing each pair of three cluster results,

by cluster analysis were further performed on LSD three STR loci [D8S1179, D7S820, and D13S317]

test in order to systematically analyze the significant probably indicated the genetic diversity among three

differences of allele frequencies [14-17].

Statistical Analysis of Genetic Diversity in 15 STR Loci from

Han, Miao and Yao Tribes in South China

and Yao. A set of symbols “-”, “+--”, “+-”, “+”, “++”, “+++”, “++++” were used to represent the levels of significant difference (< 0], (0, 0.04], (0.04, 0.08], (0.08, 0.12], (0.12, 0.16], (0.16, 0.20], (> 0.20). The “++++” and “+++” symbols focused on allele 13, 16,

17, 18 of STR loci D3S1358 and vWA interpreted a genetic diversity in Han’s D8S1179 and STR loci

(a)

[D3S1358, vWA, D18S51, D19S433] from both Miao and Yao. By comparison, symbols in two columns of

each STR locus indicated the genetic diversity between Miao and Yao. The distinctness located on 5 allele of D19S433 and allele 13 of D3S1358 and vWA.

3.2.2 LSD Analysis between Han’s D7S820 and STR Loci [CSF1PO, D5S818, D16S539, D13S317] from both Miao and Yao

(b)

The levels of significant differences about allelic with Han, focused on allele 8. CSF1PO column contained different symbols in two allelic positions of Miao and Yao probably interpreted STR diversity though not significant. Frequencies analyzed in Table 2 were not as distinct as in Table 1.

Table 1 shows that a slot located on allele 8 and

(c)

several “+” separated in allele 9, 11 and 12 of four STR

Fig. 1 The cluster analysis of Han (a), Miao (b) and Yao (c).

loci represented the genetic diversity in Han’s D7S820 The dissimilarities focused mainly on clusters [i, ii, iii], rather

and four STR loci of Miao and Yao, which had similar than iv were exactly the same among three tribes. The

hierarchy of cluster analysis with Han, focused on topologic structures in cluster iii varied between Han and Miao,

Han and Yao. allele 8. CSF1PO column contained different symbols in two allelic positions of Miao and Yao probably

3.2 Variance Analysis interpreted STR diversity though not significant. LSD values were used to discriminate the significant

3.2.3 LSD Test between Han’s D13S317 and differences of allele frequencies from specific STR loci

[CSF1PO, D5S818, D16S539, D7S820] from both [D8S1179, D7S820, and D13S317] of Han selected by

Miao and Yao

cluster analysis above between each pair of tribes. The LSD test results illustrated in Table 3

3.2.1 LSD Test between Han D8S1179 and STR represented more significant differences compared Loci [D3S1358, vWA, D18S51, D19S433] from both

with Table 2. “++++” symbols mainly located on allele Miao and Yao

8 and 12 of four STR loci consolidated the similar Since Han’s D8S1179 had the similar hierarchy of

results found in Table 2. The deviation information cluster analysis to Miao and Yao’s STR loci [D3S1358,

combining Table 2 and Table 3 suggested the most vWA, D18S51, D19S433], the result in Table 1

distinctive genetic character between Han [D13S317, revealed the significant differences allele frequencies

D7S820] and [CSF1PO, D5S818, D16S539, D7S820] ranging from allele 10 to 18 in four STR loci of Miao

from Miao and Yao, focused on allele [8, 11, 12].

Statistical Analysis of Genetic Diversity in 15 STR Loci from

Han, Miao and Yao Tribes in South China

Table 1 Han’s D8S1179 with Miao and Yao [D3S1358,

4. Discussion

vWA, D18S51, D19S433] LSD test.

D3S1358 vWA D18S51 D19S433 Huge number of STR loci in unrelated three tribal Allele Miao Yao Miao Yao Miao Yao Miao Yao

populations of South China has been typed in order to 10 + + + + + + + +

obtain genetic characteristics of these three tribes. 11 +- +- +- +- +- +- +- +-

12 + + + + +-- +-- +- +- Cluster analysis and LSD test have been applied to

13 +++ ++ +++ ++ +-- +-- + + discover several genetic hypotheses about the tribal

differentiation. The most distinctive feature of present 14 ++ + ++ +- - - - +--

13.2 +--

study is the genetic diversity among three tribes and

15 +-- +- + + +-- - ++ several regions of allelic significant [17-20].

The profound conclusions obtained from present 16 ++++ ++++ +-- + +- +- +-- +-

study include several points below.

The cluster analysis indicates an exactly identical 17 ++++ ++++ ++++ +++ - +- --

16.2 +--

+--

cluster ⅳ of each tribal population in both members 18 +- +- +++ +++ +-- +--

and topological structure. Four STR loci [D2S1138, 19 - - - +- +-- -

FGA, D21S11, TH01] included in cluster ⅳ indicate 20 - - - - +-- -

“-”, “+--”, “+-”, “+”, “++”, “+++”, “++++” represent the levels high consistent genetic characters.

of significant difference (- ∞, 0], (0, 0.04], (0.04, 0.08], (0.08, Members in clusters [iii, i] vary in three tribal 0.12], (0.12, 0.16], (0.16, 0.20], (0.20, ∞+), LSD (Han and

populations, also have the same of topological structure. Miao) = 0.032820, LSD (Han and Yao) = 0.032858.

The hierarchical positions of the three STR loci

Table 2 Han’s D7S820 with Miao and Yao [CSF1PO,

[D8S1179, D7S820, and D13S317] vary among three

D5S818, D16S539 and D7S820].

tribes according to cluster analysis. CSF1PO D5S818 D16S539 D13S317 Allele

The statistical analysis reveals the variance between Miao Yao Miao Yao Miao Yao Miao Yao

Han’s D8S1179 and clusters ⅲ [D3S1358, vWA, 7 --------

D18S51, D19S433] in Miao and Yao focuses on allele 8 +- +- +- +- +- +- +- +- [13, 16, 17, 18] of D3S1358 and vWA. 9 - - - - +- +- --

10 - - - - - - - - The variance between Han’s [D13S317, D7S820]

11 +- - - - - - - +-- and cluster i [CSF1PO, D5S818, D16S539, D7S820] in 12 ++ +- - - - - +-- +--

Miao and Yao focuses on allele [8, 11, 12]. 13 - - - - - - - -

The weightiness of STR loci in cluster ⅳ could be LSD (Han and Miao) = 0.0882, LSD (Han and Yao) = 0.0969.

omitted or reduced since the indistinguishable genetic diversity was in paternity testing or personal identify

Table 3 Han D13S317 with Miao and Yao [CSF1PO, D5S818, D16S539 and D7S820].

among these three tribes.

CSF1PO D5S818 D16S539 D7S820 The weightiness of cluster [iii, i] supposes to be Allele

Miao Yao Miao Yao Miao Yao Miao Yao aggravated because of the high degree of genetic 7--------

Acknowledgment

10 -------- 11 - - - +-- --+-

The authors appreciate all blood donors in this study. 12 ++++ ++ +- +-- +- - - +--

Without their cooperation, the study could not be 13 - - +-- -----

accomplished. This work was supported by National LSD (Han and Miao) = 0.0908, LSD (Han and Yao) = 0.0937.

Natural Science Foundation of China (No. 60736028)

Statistical Analysis of Genetic Diversity in 15 STR Loci from

Han, Miao and Yao Tribes in South China

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Journal of Life Sciences 5 (2011) 682-689

CCR5-CCR2 Gene Polymorphisms in Ethiopian Jews: Population Divergence and Its Relevance to HIV-1 Infection Resistance

1 2 3 Michael Korostishevsky 4 , Batsheva Bonne-Tamir , Zvi Bentwich , Alexander Kalinkovich and Alexander Tsimanis 5

1. Department of Anatomy and Anthropology, Tel Aviv University, Tel Aviv 69978, Israel 2. Department of Human Molecular Genetics & Biochemistry, Tel Aviv University, Tel Aviv 69978, Israel 3. Rosetta Genomics, Rehovot 76706, Israel 4. Department of Immunology, Weizmann Institute of Science, Rehovot 76100, Israel 5. Biona Ltd, Rehovot 76120, Israel

Received: January 25, 2011 / Accepted: May 18, 2011 / Published: September 30, 2011.

Abstract: CCR5 and CCR2 genes have been implicated in HIV disease progression and HIV resistance in various human populations but not in the Ethiopian Jews. The authors examined polymorphisms in the CCR5-CCR2 gene region in two groups of Ethiopian Jews,

29 non-exposed and 13 exposed but uninfected individuals. Prevalence of the CCR2-V64I and CCR5-32 mutations as well as genetic variations in the CCR5 promoter region at positions 208, 627, 676 and 927 has been studied. The authors confirmed the absence of CCR5-32 mutation in all individuals studied. Three CCR5 single-nucleotide polymorphisms (SNPs) G208T, T627C and A676G were in tight linkage disequilibrium (LD) with each other. In contrast, a lack of LD was observed across the above-mentioned SNPs and proximal SNPs C927T and distal CCR2-G190A. Only four CCR5 haplotypes - HHA, HHC, HHE and HHF*2 were identified in both

groups. Using multi-SNP analysis, no significant differences in the genotype frequencies between the groups were found ( 2 χ 3df = 4.66, P = 0.198). Observed deviation in a single SNP allele frequency (T627C SNP: 2 χ 1df = 4.14, P = 0.042) was not preserved after the Bonferroni correction. Allelic frequencies were compared to other geographically targeted worldwide populations, where clear distinction between Ethiopian Jews and Africans has been found. These data were reflected in the phylogenetic tree, in which Ethiopian Jews branch with Asians.

Key words: CCR5 and CCR2 polymorphism, Ethiopian Jews, HIV-1 infection resistance.

1. Introduction of CCR5 (CCR5-32) and a single conservative valine-to-isoleucine (V64I) mutation in CCR2 coding

The observation that chemokine receptors are used region (CCR2-G190A) have been identified. These by HIV as coreceptor for the cellular entry led to the highly specific polymorphisms are distributed through discovery of genetic factors that can affect susceptibility the world population with differing frequencies to infection with HIV or the rate of progression to dependent on ethnic groups and any particular disease once infection is established [1, 2]. Functionally population group contains a distinctive set of haplotype important polymorphisms in the regulatory region of pair combinations. The finding that polymorphisms in CCR5 gene, a 32-base pair deletion in the coding part the promoter region of CCR5 gene are associated with

differential HIV-1 disease progression suggests that Corresponding author: Michael Korostishevsky, Ph.D., senior scientist, research field: genetic epidemiology. E-mail:

these haplotypes are not functionally similar [3-7]. korost@post.tau.ac.il.

CCR5-CCR2 Gene Polymorphisms in Ethiopian Jews:

Population Divergence and Its Relevance to HIV-1 Infection Resistance

CCR5-32 deletion is the best characterized genetic of CCR2 gene. The authors estimated the magnitude of trait. In the epidemiological studies, the allelic frequency

linkage disequilibrium between the SNPs and of the deletion was 10%-20% among Caucasians,

performed multi-SNP analysis between the samples. particularly amongst those of Northen European descent

Using the CCR5 haplotype distribution data for with 1% homozygosity. This mutation is extremely rare

different ethnic groups, the authors investigated the in African and Asian population [8, 9]. Individuals

phylogenetic relationships for Ethiopian Jews. homozygous for CCR5-32 mutation are almost

2. Materials and Methods

completely resistant to HIV infection most probably due to lack of CCR5 receptor on their cell surfaces [10].

2.1 Subjects

Studies of CCR5-32 mutation in exposed but uninfected

A total of 29 HIV-1-negative (control) and 13 individuals have revealed that only a small proportion of exposed but uninfected seronegative (ESN) Ethiopian them were homozygous for this mutation [11]. Jews were sampled for this study. The evaluation of Heterozygosity for CCR5-32 mutation is associated

clinical status of individuals including the presence of with delayed progression to AIDS in infected

antibodies to HIV-1 and HIV-1 viral particles has been individuals. Moreover, frequency of heterozygosity is

performed in the Kaplan Medical Center, Rehovot, significantly higher in long term non-progressors than in

Israel, as described [17]. Samples were coded and progressors and rapid progressors [8-10, 12, 13]. The

mechanism of protection is not clear and it is believed tested blind. Informed consent was obtained for the that CCR5 expression may be altered in these

collected samples.

individuals. CCR2-V64I mutation is associated with

2.2 Genotyping

delay in progression to AIDS, probably due to the heterodimerization and sequestration of the CCR5

The authors verified the presence of CCR5- Δ32 receptor [14, 15].

deletion and genotyped sequence variations for the Our research focused on CCR5 genetics of Ethiopian

single-nucleotide polymorphism (SNP) G208T, T627C, Jews currently living in Israel. This population

A676G and C927T in the CCR5 promoter region as originated in the north of Lake Tana in Gondar,

well as G190A mutation in the coding part of CCR2 Ethiopia, and several thousands of them were airlifted

gene. The authors used genomic DNA obtained from to Israel first during the Ethiopian civil war (1984-1985)

peripheral blood lymphocytes. The DNA samples were and then in 1989.

subjected to a polymerase chain reaction-restriction The purpose of this study was to examine whether

fragment length polymorphism (PCR-RFLP) assay as the DNA polymorphisms at the loci that encode CCR5

previously described [12, 18, 19]. PCR amplification and CCR2 receptors, can potentially explain the

was performed to amplify CCR5 promoter and CCR5 persistent seronegativity in a group of exposed

and CCR2 genes fragments covering the polymorphic seronegative (ESN) individuals. Using sites (Table 1). evolutionary-based CCR5 haplotype classification [3,

For CCR2-V64I mutation, the PCR products were 16], the authors have characterized this polymorphism

digested with FokI. After digestion, the products of in two groups of Ethiopian Jews: Healthy individuals

digestion for CCR2 and the PCR products for delta-32 without any history of HIV infection or ESN

CCR5 were loaded on a 4% agarose gel in 0.5 × TBE individuals. The authors assessed of CCR5-32 deletion

and electrophoresed at 85 V for 2 hours. as well as several single nucleotide polymorphisms

CCR5 promoter polymorphism was also detected by (SNPs) in the CCR5 promoter region and coding part

PCR amplification using [- 32 P]-dATP. PCR products

CCR5-CCR2 Gene Polymorphisms in Ethiopian Jews: Population Divergence and Its Relevance to HIV-1 Infection Resistance

Table 1 SNPs and primer to PCR-amplify the CCR5 and CCR2 genes.

G190A, CCR2 ORF

5’GAC AGA AGC AAA CAC AGC CA3’ G208T, CCR5 Exon1

F 5’ACG GTG CTC CCT GTC ATA AA3’

F9 5’GAT TCT GTG TAG TGG GAT GAG C3’

5’GAG TTT CTT GTA GGG GAA CGG3’ T627C, CCR5 Exon2

A676G, CCR5 Exon2

F8 5’CTC ATC TGG CCA GAA GAG CTG3’ C927T, CCR5 Exon3

5’ACT GTG ACC CTT TCC TTA TC3’

CCR5- Δ32 ORF

62,036-62,067

5A 5’AGG TCT TCA TTA CAC CTG CAG C3’ 5B 5’CCT CTC ATT TCG ACA CCG AAG C3’

*The numbering of nucleotide positions is based on GenBank sequence U95626.

were resolved by electrophoresis in 6.5% that amplified the region encoding the 32-bp deletion. polyacrylamide gel.

CCR5-32 deletion was not detected in either the control or ESN groups, indicating that this mutant allele is

2.3 Statistical Analysis probably rare or absent in the Ethiopian Jewish

Genotype and allele frequencies of the SNPs were population. CCR5-32 allele is very common in white calculated by direct counting. Possible differences in

populations, yet this allele is rare in people of African the frequencies of each of the SNP genotypes and

and Asian descent including Ethiopian Jews [30]. alleles between the samples were estimated using the 2 χ The results of LD tests between pairs of SNP markers

test, as described elsewhere [20, 21]. The Arlequin for ESN and control groups are depicted in Fig. 1. As software package [22] was used for: 1) Evaluation of

can be seen, three internal SNPs, CCR5-G208T, genetic distances between different populations [23]; 2)

CCR5-T627C and CCR5-A676G, demonstrated Estimation of pairwise LD between the SNP markers

significant linkage disequilibrium (P < 0.05). Distal [24, 25]; 3) Detection of departure from SNP CCR2-G190A and proximal CCR5-C927T were Hardy-Weinberg equilibrium (HWE) [26]; and 4)

found in strong linkage disequilibrium (P < 0.0005), Calculation of the maximum likelihood (ML) of

while a lack of linkage disequilibrium between them haplotype frequencies [27].

and the internal SNPs was observed. Our results Based on the ML haplotype frequency estimates, the

confirmed the strong linkage disequilibrium between likelihood ratio test (LRT) for sample differentiation

CCR2-64I and CCR5-927T, which had been reported was evaluated as previously described [28]. PHYLIP

previously [31].

software package [29] was used for phylogeny Allelic and genotype frequencies for the CCR2-G190A and four CCR5 promoter

inferences based on the CCR2-CCR5 region genetic polymorphisms among the control and ESN

distances. The Bonferroni corrections were performed

individuals are shown in Table 2.

by the SISA online procedure

A discrepancy in allelic frequency between the (http://home.clara.net/sisa/bonfer.htm).

control and ESN individuals was found for the

3. Results and Discussion 2 CCR5-A676G SNP ( χ

1df = 4.14, P = 0.042). The association suggests that this mutation may be tracking

3.1 Genotype and Allele Frequencies the same effect on HIV-1 disease progression.

Both the control and ESN groups were genotyped for However, this deviation of one from five SNPs typed in the CCR5-32, CCR2-G190A and CCR5 promoter

this study is not preserved after the Bonferroni alleles constituting the CCR5 human haplotypes.

correction, which takes into account multiple-test The authors performed DNA PCR by use of primers

problem.

CCR5-CCR2 Gene Polymorphisms in Ethiopian Jews:

Population Divergence and Its Relevance to HIV-1 Infection Resistance

SNP

V64I G208T T627C A676G C927T

V64I G208T T627C

A676G C927T

Fig. 1 Significance level for pairwise LD in the ESN and control samples. LD significance above the diagonal corresponds to the ESN sample and below the diagonal to the control sample (white: P > 0.05; gray: 0.005 < P < 0.05; dark gray: 0.0005 < P < 0.005; black: P < 0.0005).

Table 2 Genotype and allele frequencies of each SNP 1 in ESN and control samples.

SNPs Genotype distribution Allele distribution

2 χ 2 test Allele ESN Control χ test CCR2-64I

ID 2 Position 3 Genotype ESN

Control

0.40 (G190A)

CCR5-G208T 58,934 GG 7 8 3.87 3.09 G 20 33

GT 6 17 TT 0 4

T6 25

CCR5-T627C 59,353 TT 1 8 4.57 4.14 T9 34

CT 7 18 CC 5 3

C 17 24

CCR5-A676G 59,402 AA 7 8 3.87 3.09 A6 33

AG 6 17 GG 0

G 20 25

CCR5-C927T 59,653 TT 1 0 2.29 0.40 C 19 46

CT 5 12 CC 7 17

T7 12

1 SNPs that were non-polymorphic in both samples are not presented in the table: CCR5-A29G (100% A) and CCR5-630 (100% C). 2 SNP designations are according to Ref. [32]. 3 The numbering of nucleotide positions is based on GenBank sequence U95626.

CCR5-CCR2 Gene Polymorphisms in Ethiopian Jews: Population Divergence and Its Relevance to HIV-1 Infection Resistance

3.2 Haplotype Polymorphism pairs was HHC/HHE (23.1%), followed by HHC/HHF*2, HHE/HHE and HHE/HHF*2, the

Only four haplotypes among known CCR5 haplotypes that were the most common pairs. Such haplotypes were detected in ESN and control distribution of haplotype pairs, both in the control and individuals (Table 3). ESN groups are typical of Caucasians, whereas two The haplotypes are notated according to the pairs, HHA/HHA and HHA/HHF*2 haplotypes, evolutionary-based classification of the CCR5 [3, 16]. typical of Africans [32] were rare in Ethiopian Jews. The HHF*2 frequency in the ESN individuals is It has been previously shown that polymorphism in slightly higher than that in the control group (26.9% vs. the CCR5 promoter region is involved in HIV-1 20.7%), although the difference did not attain statistical infection, disease progression, and disease transmission significance. In the control group, the most common [12, 14, 33, 34]. However, comparative analysis of CCR5 haplotype was HHC (43.1%), whereas among CCR5 polymorphisms undertaken in ESN individuals ESN individuals, the most common haplotype was for three ethnic groups (Caucasian, Asian and African) HHE (38.6%). Of note, both haplotypes have significantly revealed the controversial results. Mangano et al. higher frequencies in Caucasians [3]. The minor examined 649 HIV-1-exposed Argentinean children at haplotype in both studied groups was HHA (15.5% and risk for parental infection, and found that the frequency 11.5%, respectively), which is more frequent in of HHE-containing haplotypes was higher in the

Africans. However, the multi-SNP likelihood ratio test HIV-positive children. Moreover, homozygosity and

did not find significant differences between the studied

heterozygosity of HHE haplotype have been suggested groups ( χ 3df = 4.66, P = 0.198). Interestingly, as major factors associated with an increased risk of

genotyping of samples from both studied groups failed parental infection whereas delay in progression to AIDS

to detect presence of HHD and/or HHB haplotypes, which are specific to African population [3, 16].

is associated with HHF*2 haplotype [35]. Gonzalez et al. have found that HHE haplotype influenced the

Eight different genotypes were found in the control outcome of European Americans adults [3]. Although

group: HHA/HHA, HHA/HHC, HHA/HHF*2, HHF*2 and HHE haplotypes are associated with the

HHA/HHE, HHC/HHC, HHC/HHE, HHC/HHF*2, opposite effects, both haplotypes are carriers of

HHE/HHF*2. In the ESN group seven haplotype pair “non-protective” 303-a allele and in the luciferase assay

combinations, namely HHA/HHC, HHA/HHF*2, exhibit the highest transcriptional activity [16]. HHF*2

HHA/HHE, HHC/HHE, HHC/HHF*2, HHE/HHE and homozygosity was also associated with HIV-1

HHF*2/HHF*2 were detected. In the control group, the resistance in discordant couple cohort from Thailand. most prevalent haplotype pairs were HHC/HHF2 Furthermore, in vitro infection experiments showed that (27.6%), followed by HHC/HHC and HHC/HHE. On PBMC isolated from the HIV-1-exposed and the other hand, in the ESN group the leading haplotype unexposed seronegative women carrying different

Table 3 ML estimates of haplotype frequencies in ESN and

CCR5 haplogroups (including HHF*2/HHF*2) had no

control samples.

differences in susceptibility to HIV-1 infection [36, 37]. Haplotype HH ESN Control OR LRT*(P-value)

A cohort study of Ugandan population showed no G-G-C-A-C HHE 0.385 0.207 1.859 association between CCR5 polymorphisms and the rate G-T-T-G-C HHC 0.231 0.431 0.535

A-G-C-A-T HHF*2 0.269 0.207 1.301 of disease progression [38]. Polymorphisms in the

G-G-T-A-C HHA 0.115 0.155 0.744 CCR5 promoter region may influence cell surface * The likelihood ratio test (LRT) for sample differentiation was

expression and consequently could influence individual evaluated as previously described [28].

susceptibility to HIV [39]. However, similar expression

CCR5-CCR2 Gene Polymorphisms in Ethiopian Jews:

Population Divergence and Its Relevance to HIV-1 Infection Resistance

level of CCR5 was found in HIV-exposed uninfected

3.3 Population Divergence

females and unexposed controls from Kenya and Using the CCR5-CCR2 haplotype frequency Ethiopia [40, 41]. Taken together, these data suggest estimates, the genetic distances (Corrected Average that the distribution of polymorphisms in CCR5 Pairwise Difference: ARLEQUIN software package) promoter region varies significantly across between Ethiopian Jews and other 7 populations were race/ethnicity groups, so that the same mutation in

evaluated (Table 4).

different race groups may have various or even opposite The population relationship reconstructed by using

functional effects. these distances (the UPGMA algorithm: PHYLIP

Study of the genetic structure of Ethiopian Jews by software package) was also undertaken and the results

using mtDNA and some nonrecombinant Y-chromosome markers and 5’-globin haplotypes

are depicted in Fig. 2.

elements clearly demonstrated that Ethiopian Jews are As can be seen, the dendrogram shows two

a mixture of African and Caucasian (Asian) population well-defined population groups: The first one and are significantly different from other Jewish

containing two populations, namely Non-Pygmy and communities [42-46]. Several authors argued that

American Africans, and the second one containing the Ethiopian Jews derive mostly from Africans [47];

remaining six populations, which are further divided. however, both cultural and historic evidence shows

Within these groups, Ethiopian Jews are “sisters” to tight relationship between populations of Ethiopia and

Asian populations (non-Indians and Indians), while Asia (Near East and southern Arabia).

Thai population is found to be the most distant.

Table 4 Population average pairwise differences*.

Population Non-pygmy Afr. Amer European Non-indian Indian Eth. Jews Thai Argentina Non-pygmy # 0.78 0.79 0.83 0.85 0.83 0.85 0.87 0.83 Afr. Amer # 0.21 0.79 0.82 0.81 0.82 0.83 0.81 0.81 European # 11.04 7.26 0.66 0.71 0.71 0.72 0.7 0.73 Non-indian # 11.07 7.72 3.14 0.7

0.73 0.71 0.66 0.73 Indian # 7.44 4.55 1.49 1.56 0.73 0.73 0.7 0.75 Eth. Jews

10 6.92 3.64 0.16 0.41 0.72 0.66 0.74 Thai ##

19.36 15.17 8.03 2.75 4.91 1.77 0.57 0.7 Argentina # 6.5 3.94 2.45 0.88 0.99 1.09 4.27 0.75 *Above diagonal: Average number of pairwise differences between populations, d(ij).

Diagonal elements: Average number of pairwise differences within population, d(i). Below diagonal: Corrected average pairwise difference, d(i, j)-(d(i)+d(j))/2. Row data of CCR5 haplotypes distribution was taken from: # - [32] and ## - [37].

NON-PYGMY

4. Conclusion

AFR. AMER.

EUROPEAN

No statistically significant differences between HIV-

NON-INDIAN

unexposed and exposed uninfected Ethiopian Jews at

ETH. JEWS

the allele, genotype, or haplotype level have been found.

INDIAN ARGENTINA

The pattern of CCR5-CCR2 genetic variations in Eth-

iopian Jews resembles the one found in Asian populations

THAI

Fig. 2 UPGMA tree based on CCR5 haplotype frequencies.

and is distinguished from that found in Africans.

CCR5-CCR2 Gene Polymorphisms in Ethiopian Jews: Population Divergence and Its Relevance to HIV-1 Infection Resistance

Acknoledgments

HIV-1 infection of CD4 lymphocytes from persons who remain uninfected despite multiple high-risk sexual

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