Distribution of Killer Cell Immunoglobulin-Like Receptors in the Macedonian
Population
Eli Djulejica, Dejan Trajkova, Aleksandar Petlichkovskia, Ana
Strezovaa, Slavica
Hristomanovaa, Derek Middletonb, Mirko Spiroskia, *
aInstitute
of Immunobiology and Human Genetics, Faculty of Medicine, University
"Ss. Kiril and Metodij", Skopje, Republic of Macedonia; bRoyal Liverpool University
Hospital and School of Infection and Defence, Liverpool University, Liverpool, L7
8XP, United Kingdom
*Correspondence to:
Mirko Spiroski, MD, PhD
Institute of Immunobiology and Human Genetics,
Faculty of Medicine, University "Ss. Kiril and Metodij",
1109 Skopje, PO Box 60, Republic of Macedonia
Tel.: +389-2-3110556
Fax: +389-2-3110558
URL: http://www.iibhg.ukim.edu.mk/
e-mail: mspiroski@yahoo.com
Short title: KIR in Macedonians
1
Abstract
The aim of this study was to analyze Killer Ig-Like Receptor (KIR) Gene
Polymorphism in the Macedonian Population. The studied sample consists of 214
healthy unrelated individuals, aged 20-35 years. All individuals are of Macedonian
origin and nationality, residents of different geographical regions. The spoken
language is Macedonian. Blood samples were collected after written consent, DNA
was isolated from peripheral blood leukocytes by the phenol-chlorophorm extraction
method. PCR-based method designed to detect the presence and absence of KIR
genes was used (The PEL-FREEZ KIR Genotyping SSP Kit). The population
genetics analysis package, Arlequin, was used for analysis of the data. We found
that all 16 KIR genes were observed in the Macedonian population and framework
genes 3DL3, 2DL4, and 3DL2 were present in all individuals. Total of 56 different
KIR genotypes were found to be present in Macedonian population, based on the
presence of 16 KIR genes. Neighbor-Joining phylogenetic tree, constructed on the
basis of standard genetic distances of KIR genes, shows that Macedonian
population
is
in
the
same
BrazilSouthEastCaucasian,
cluster
with
EnglandWestMidlandsIndianAsian,
RomaniCaucasians,
Basque,
EnglandWestMidlandsCaucasian, Reunion, and SpainGranada populations. The
frequency of KIR loci in Macedonian population shares several general features with
other Caucasoid populations studied before.
Key words: Killer immunoglobulin-like receptor (KIR) gene polymorphism, genetic
distance, KIR genotyping, PCR-SSP, Macedonian population
2
1. Introduction
Killer immunoglobulin-like receptors (KIR) are cell surface molecules
important in the regulation of activity of the natural killer (NK) cells and some T cells
in either inhibitory or activating manner [1]. KIR molecules are encoded by the KIR
gene family clustered within the leukocyte receptor complex on chromosome
19q13.4 [2, 3]. KIR genes exhibit allelic, haplotypic, and gene content variability [47]. KIR haplotypes differ in the number and kind of KIR genes they contain. Thus,
KIR haplotypes can be distinguished according to their gene content. Because the
individual KIR genes are polymorphic, KIR haplotypes that are identical by gene
content can be further subdivided according to allele combination. Diversity in
human KIR genotype therefore is sum from three components: haplotypic gene
content, allelic polymorphism and the combination of maternal and paternal
haplotypes. The combined effects of these three components are such that
unrelated individuals usually differ in KIR genotype and ethnic populations have
widely differing KIR genotype frequencies.
In general, two KIR haplotypes are recognized: A and B [7-9]. The latest
haplotype definition (14th International HLA and Immunogenetics Workshop, 2005)
identifies haplotype A to be composed of KIR3DL3, KIR2DL3, KIR2DP1, KIR2DL1,
KIR3DP1, KIR2DL4, KIR3DL1, KIR2DS4, and KIR3DL2 genes, while all other
haplotypes are described as haplotype B.
With the exception of null alleles and possibly 3DL3, all known KIR-s in a
given individual's KIR gene repertoire are expressed [1-12]. In heterozygous
individuals, NK clones can express none, one or both alleles of 3DL1 and 3DL2, but
usually both alleles of 2DL4 [13].
3
Population studies have revealed that KIR gene frequencies and genotype
content vary considerably among different ethnic groups, and that frequencies of A
and B haplotypes have also been found to differ between populations [4, 7, 14-22].
Results from population studies to date indicate the extent of KIR gene diversity;
some of these studies have also shown the association of the presence or absence
of specific KIR genes in certain human diseases [23-27].
Although comparisons with the major histocompatibility complex (MHC) are
obvious, what is less clear are the functional implications of KIR diversity and
whether it serves us a useful purpose [28, 29].
Macedonian population is of special interest for anthropological studying in
the light of opened questions regarding its origin and the migrations which resulted in
settlement of Macedonians in the heart of Balkan Peninsula. Macedonia is located in
the Central Balkans, bordering Bulgaria, Greece, Albania, Serbia and Kosovo
province, covering an area of 25.710 kms. According to the 2002 census, the
country’s population was 2.022.577. Data on the declared ethnic affiliation from the
2002 census reported that 64.1% of the population identify themselves as
Macedonian, 25.17% as Albanian, 3.95% as Turks, 2.66% as Roma, 1.78% as
Serbs, 0.84% as Bosniacs, 0.48% as Vlachs and 1.04% others [30]. The country
seceded peacefully from Yugoslavia after an independence referendum, held in
September 1991.
This is the first study of the diversity of KIR genes in Macedonian populations.
The aim of this study was to determine the frequencies of 16 KIR genes and
pseudogenes (KIR2DL1, KIR2DL2, KIR2DL3, KIR2DL4, KIR2DL5, KIR3DL1,
KIR3DL2, KIR3DL3, KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS4, KIR2DS5, KIR3DS1,
KIR2DP1, and KIR3DP1), genotypes and to compare them with populations for
4
which similar data are available. Finally, genetic distances between Macedonian and
other compared populations were calculated.
2. Material and Methods
2.1. Population samples
The study included 214 unrelated healthy individuals, all Macedonians of
Macedonian origin and nationality, and residents of different regions of the Republic
of Macedonia. Each individual was interviewed on a one-to-one basis, his/her
genealogy was recorded for the last three generations, and a signed consent was
obtained. Admixture, if any, was recorded for each individual. Individuals with only
one Macedonian parent were excluded from the study. After signing of written
consent, genomic DNA was extracted from the peripheral blood leukocytes using
standard phenol/chloroform procedure, described elsewhere [31], and stored in the
anthropology project field of the Macedonian Human DNA Bank (hDNAMKD) [32]
until processing.
2.2. PCR amplification
For KIR genotyping commercially available PEL-FREEZ KIR genotyping SSP
kit (Dynal Biotech, Brown Deer, WI) was used. It is a PCR-based method (using
sequence-specific priming approach) designed to detect the presence and/or
absence of 16 KIR genes and pseudogenes defined by the International
nomenclature committee of WHO [33, 34]. Briefly, locus specific primer sets,
dispensed in a 96 well thermal tray were used for amplification of genomic DNA.
After mixing a reaction buffer with a human genomic DNA sample and Taq DNA
polymerase, the mixture was dispensed to the tray, sealed and then thermally cycled
5
as follows: (1 min at 95, 30 cycles of 94 for 20 seconds, 63 for 20 seconds and
72 for 90 seconds). After the amplification, the PCR products are loaded and
separated by electrophoresis onto a 2% agarose gel stained with ethidium bromide,
after which the results are interpreted using a worksheet for the specific amplification
patterns. The presence of each KIR gene was determined by the presence of a band
of DNA of the expected size. All PCRs contained an internal positive control
consisting of an additional pair of primers specific for the growth hormone gene [35],
and a negative control. Individuals were determined negative for a particular KIR
gene when a band of expected size was absent in the presence of a band for the GH
gene.
2.3. Statistical analysis
The occurrence of KIR genes in individuals (F) was obtained by direct
counting. Gene frequencies (GF) were calculated using the formula F=1-(1-F). For
analysis of the molecular polymorphism of the locus studied, the Arlequin software
version 3.0 [36] (Genetics and Biometry Laboratory, University of Geneva,
Switzerland) was used. The Hardy-Weinberg equilibrium [37] and the EwansWatterson [38] statistics were calculated in order to examine the presence of
selective forces influencing the allele distribution. The percentage of individuals
carrying each KIR gene was also calculated. Linkage disequilibrium (LD) values for
two locus associations were calculated using 2×2 tables [39]. Because LD is not
independent of allele frequencies, normalized LD was calculated as described
previously [40, 41]. The statistical significance of LD values was assessed by Yates
X2 analysis on the corresponding 2×2 contingency table. A neighbour-joining tree
6
was constructed from Nei’s genetic distances using the PHYLIP phylogeny inference
package [42].
Group nomenclature (AA, AB, BB) of KIR was defined if any of genes 2DL2,
2DL5, 3DS1, 2DS1, 2DS2, 2DS3, 2DS5 are present, genotype is taken as having B.
If
none
of
these
are
present
genotype
is
considered
as
AA.
If any are present and 3DL1, 2DL1, 2DL3 and 2DS4 are all present then genotype is
considered as AB. However, if any of 3DL1, 2DL1, 2DL3, 2DS4 genes are missing,
genotype is considered as BB. KIR genotypes were numerated according the KIR
Database >> Genotype Reference [43].
For comparison of KIR gene frequencies and genetic distance analysis, we
have used data for KIR genotypic polymorphisms published at the Allelefrequencies
database (www.allelefrequencies.net) [44]. The number of KIR genes allocated at
this database differs for different populations, depending on the typing method used.
We have included in our study and used for comparison only those having complete
data for 14 different genes. According to this criterion, 33 populations were selected
out of total of 82 populations.
3. Results
3.1. KIR gene frequencies
The presence and absence of the 16 KIR genes (14 genes and 2
pseudogenes) determined in the 214 healthy individuals, randomly selected from the
Macedonian population are shown in Table 1. All 16 KIR genes were observed in the
Macedonian population and framework genes 3DL3, 2DL4, and 3DL2 were present
in all individuals. The most frequently present KIR genes were: 2DL1 (0.94), 2DL2
(0.59), 2DL3 (0.89), 2DL5 (0.41), 3DL1 (0.93), 2DP1 (0.98), 2DS1 (0.48), 2DS2
7
(0.56), 2DS3 (0.36), 2DS4 (0.94), 2DS5 (0.30), 3DS1 (0.39), 3DP1 (0.99),
respectively. Most of individuals (99.5%) had 3DP1; 99% had 3DP1*003, and 24%
had 3DP1*001/002. In 41% of individuals we found 2DL5; 28% had 2DL5A*001, and
25% had 2DL5B*002/004. In 94% of individuals we found 2DS4; 25% had
2DS4*001-002, and 85.9% had 2DS4*003 (Table 1).
<Table 1>
3.2. Genotype frequencies
KIR genotypes, groups, genotype ID, number of individuals and percentage of
distribution is given in Fig. 1. Total of 56 different KIR genotypes were found to be
present in studied population, based on the presence of 16 KIR genes. We found
one group AA, genotype ID 1 in 46 individuals (21.5%), 33 group AB, and 22 group
BB genotypes. The most frequent genotypes in group AB are genotypes ID 4 with 27
individuals (12.62%) and ID 2 with 11 individuals (5.14%). The most frequent
genotypes in group BB are ID 73 (2.34%) and ID 71 (1.87%). We found 5 new
genotypes in AB group and 9 new genotypes in group BB which are referred in KIR
database of Allelefrequencies.net (Fig. 1).
<Figure 1>
3.3. Linkage disequilibrium
The
classical
linkage
disequilibrium
coefficient
(D),
linkage
disequilibrium coefficient D standardized by the maximum value it can
take (Dmax), given the allele frequencies (D’), standardised simple
measure of linkage disequilibrium (r2), and statistical significance (P) for
KIR genes are shown in Table 2. The genes present in all individuals were
excluded from the analysis.
<Table 2>
8
Pair of KIR loci that displayed significant (P<0.05) LD in Macedonian
population are given in Table 3. From the Table 3 we can see positive and negative
LD with KIR genes.
<Table 3>
3.4. Genetic distances and phylogenetic tree
For comparison of KIR gene frequencies and genetic distance analysis, we
have used data for KIR genotypic polymorphisms published at the Allelefrequencies
database (www.allelefrequencies.net) [44]. The number of KIR genes allocated at
this database differs for different populations, depending on the typing method used.
We have included in our study and used for comparison only those having complete
data for 14 different genes. According to this criterion, 33 populations were selected
out of total of 82 populations.
Diversity of investigated populations for KIR genes is given for all loci, and
locus by locus on the Table 4. Measure of population differentiation (GST) for all loci
was 0.067, for the locus 2DL3 was the biggest (0.133), and the smallest was for
locus 2DS3 (0.046). Total genetic diversity in the pooled populations (HT) for all loci
was 0.285 the biggest was for locus 2DL5 (0.440), and the smallest was for the
locus 2DL4 (0.120). Mean diversity within each population (HS) for all loci was 0.317,
the biggest was for the locus 2DL5 (0.419), and the smallest was for the locus 2DL4
(0.011). We did not found diversity for 3DL2 and 3DL3 loci (Table 4).
<Table 4>
The average heterezygosity for the different populations and their standard
error is shown on Table 5 and is around 0.2 for most of the populations. The lowest
heterezygosity is observed for Japanese population (0.152 ± 0.035), while it is
highest in PakistanKarachi (0.374 ± 0.048).
9
<Table 5>
The phylogenetic tree (Figure 2) was constructed on the basis of comparison
of gene frequencies for KIR loci in different populations. It clearly shows the relations
between the Macedonian and other compared populations. The closest genetic
distance is observed between the studied Macedonian population and the
SpainGranada
population
(SGD=0.00),
while
the
most
genetically
distant
populations were SouthAfricaXhosa (SGD=4.33) and Tarahumara (SGD=4.04)
(Table 5).
Populations from Oman, Lebanon, BelgiumKIRPop, Guadeloupe, Senegal
and SouthAfricaXhosa are in the same cluster on the bottom of the tree. On the
opposite side of the genetic tree are IndiaNorthHindus, PakistanKarachi, and
SouthAsians in oner subclaster together with the rest of populations in the same
claster
(EnglandWestMidlandsIndianAsian,
RomaniCaucasians,
Basque,
BrazilSouthEastCaucasian,
EnglandWestMidlandsCaucasian,
Reunion,
Macedonia, and SpainGranada). The rest of populations are in the middle clasters
(Fig. 2).
<Figure 2>
4. Discussion
The present study has been initiated to define reliable estimates of the KIR
gene frequencies for the Macedonian population suitable for use in the
anthropological studies in order to better understand genetic distances, migrations
and relations between different populations and ethnic groups.
We found that all 16 KIR genes were observed in the Macedonian population
and framework genes 3DL3, 2DL4, and 3DL2 were present in all individuals. The
most frequently present KIR genes were: 2DL1 (0.94), 2DL2 (0.59), 2DL3 (0.89),
10
2DL5 (0.41), 3DL1 (0.93), 2DP1 (0.98), 2DS1 (0.48), 2DS2 (0.56), 2DS3 (0.36),
2DS4 (0.94), 2DS5 (0.30), 3DS1 (0.39), 3DP1 (0.99), respectively.
There are numerous publications of KIR gene distribution in different
populations, but the most comprehensive is allelefrequencies.net database [44].
Two-dimensional plot from multidimensional scaling analysis based on pair wise RST
values from KIR genotypes have shown that Poles showed close clustering with
German [45], British [15], Greek [19] and Australian Caucasoid [9]. Northern Irish
Caucasoid [46] and Palestinians [15] were somewhat further away. Other
populations such as Asiatic Indians [17], Pakistanis from Pakistan, Pakistanis from
Trinidad, Africans from Trinidad [16] and Thais [15] were even more distant, as
expected. Interestingly, a Californian American sample that was the first human
population described for KIR genotypes is found to be very distant from all other
populations [8]. Finally, Koreans [20] and Japanese [8] were relatively close together
and far away from all other groups [47].
Total of 56 different KIR genotypes were found to be present in Macedonian
population, based on the presence of 16 KIR genes. We found one group AA, 33
group AB, and 22 group BB genotypes. The most frequent genotypes in group AB
are genotypes ID 4 and ID 2. The most frequent genotypes in group BB are ID 73
and ID 71. We found 5 new genotypes in AB group and 9 new genotypes in group
BB which are included in KIR database of Allelefrequencies.net [44].
Haplotype A was found to be predominant in all four Pacific Islands
populations and has previously been shown to be prevalent in Caucasian, Korean,
Thai, and Japanese populations, while haplotype B has been shown to be more
frequent in North Indians, Palestinians, South Asians, and Afro-Caribbeans [4, 7, 1422]. This data also correlate with distances observed in the neighbour-joining tree
11
where Pacific Islands populations were grouped with other, predominantly haplotype
A, populations and away from predominantly haplotype B populations.
In general, an understanding of which markers are in strong linkage
disequilibrium allows for the more rational design of genetic studies. In the KIR
system in particular, where genes of different groups may act inhibitory or
stimulatory and in combination with HLA ligands, knowledge of the existing
disequilibria is vital to our understanding of which gene combinations are important
in disease.
Most KIR genes were present in similar frequencies in the four Pacific Islands
populations [48], and comparable KIR polymorphism in Indonesian populations that
probably reflects the large geographical spread of the Indonesian archipelago has
been published [49]. Polynesians have been found to be genetically less
heterogenous based on analysis of six minisatellites, but with differences observed
in gene frequencies in geographically close populations, which have been largely
attributed to the bottleneck effect arising from small founding populations followed by
subsequent random genetic drift [50].
We report here genetic distances of 14 KIR genes for 33 populations,
deposited on the allelefrequencies.net [44], and they group in several clusters.
Populations from Oman, Lebanon, BelgiumKIRPop, Guadeloupe, Senegal and
SouthAfricaXhosa are in the same cluster on the bottom of the tree. On the opposite
side of the genetic tree are IndiaNorthHindus, PakistanKarachi, and SouthAsians in
other subcluster together with the rest of populations in the same cluster
(EnglandWestMidlandsIndianAsian, BrazilSouthEastCaucasian, RomaniCaucasians,
Basque,
EnglandWestMidlandsCaucasian,
Reunion,
Macedonia,
and
SpainGranada). The rest of populations are in the middle clusters. In the similar
12
investigation of genetic distances based on the results of 56 populations, populations
tended to group together according to a geographical gradient. Macedonian
population was in the same cluster together with SpainGranada [51], which is
identical with our investigation. Analyses based on KIR DS or KIR haplotype B
genes show that populations are related according to geography, like a good
anthropological marker (i.e.:HLA or Y chromosome systems). The results based on
KIR DL or KIR haplotype A genes do not show such a correlation [52]. We do not
have any explanation about the population positions in the clusters, but different
selective pressures in the past could be one of the explanations.
In summary, the results of KIR gene frequencies, genotype frequencies, and
linkage disequilibrium in Macedonian population are similar with the published data
for KIR distributions in Caucasoid, and can be used for anthropological comparisons,
as well for association studies with different diseases. Neighbour-Joining
phylogenetic tree, constructed on the basis of standard genetic distances of KIR
genes, shows that Macedonian population is in the same cluster
with
EnglandWestMidlandsIndianAsian, BrazilSouthEastCaucasian, RomaniCaucasians,
Basque, EnglandWestMidlandsCaucasian, Reunion, and SpainGranada populations.
Acknowledgement
The authors thank Elena Zaharieva for isolation of genomic DNA, and taking
care of the Macedonian Human DNA Bank (www.hdnamkd.org.mk). This work was
supported in part from the ICGEB collaborative research program (CRP/MAC03-01),
Trieste, Italy.
13
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Cladistics 1989;5:164–6.
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M,
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21
Table 1: Observed and estimated KIR gene frequencies for Macedonian population (N = 214).
OF
S.D. of
OF
EF
Frequencies for Macedonian population
Inhibitory KIR
2DL4
2DL5
3DL1
3DL2
3DL3
2DS1
1
0.416
0.939
1
1
0.481
Pseudogenes
2DP1
3DP1
0.98
0.995
2DL1
0.949
2DL2
0.589
2DL3
0.897
0.009
0.005
0.015
0.034
0.021
0
0.034
0.016
0
0
0.86
0.90
0.76
0.36
0.67
1
0.24
0.74
1
1
2DS2
0.565
Noninhibitory KIR
2DS3
2DS4
0.360
0.944
2DS5
0.304
3DS1
0.392
0.034
0.034
0.033
0.16
0.031
0.033
0.28
0.34
0.20
0.76
0.17
0.22
OF, observed frequency; S.D. of OF, standard deviation of observed frequency; EF, estimated frequency.
22
Table 2. LD analysis for KIR loci in Macedonian population.
2DS2
2DS2
2DL2
2DL3
2DP1
2DL1
3DP1
3DL1
3DS1
2DL5
Da
D’
r2
P
D
D’
r2
P
D
D’
r2
P
D
D’
r2
P
D
D’
r2
P
D
D’
r2
P
D
D’
r2
P
D
D’
r2
P
D
D’
r2
P
2DL2
0.2278
0.9799
0.8725
<0.0001
2DL3
-0.0447
-1.0000
0.0881
<0.0001
-0.0423
-1.0000
0.0800
<0.0001
2DP1
-0.0447
-1.0000
0.0881
<0.0001
-0.0077
-1.0000
0.0133
0.0916
0.0168
1.0000
0.1662
<0.0001
2DL1
-0.0081
-1.0000
0.0146
0.0767
-0.0165
-0.7789
0.0230
0.0266
0.0134
0.2907
0.0400
0.0034
0.0131
0.7365
0.1906
<0.0001
3DP1
-0.0177
-0.7908
0.0260
0.0182
-0.0019
-1.0000
0.0033
0.4022
0.0042
1.0000
0.0410
0.0031
0.0046
1.0000
0.2465
<0.0001
-0.0002
-1.0000
0.0003
0.8155
3DL1
-0.0020
-1.0000
0.0036
0.3795
-0.0110
-0.4388
0.0087
0.1725
0.0031
0.0569
0.0018
0.5318
-0.0011
-1.0000
0.0012
0.6076
0.0156
0.3225
0.0871
<0.0001
-0.0003
-1.0000
0.0003
0.7988
23
3DS1
-0.0077
-0.2920
0.0042
0.3409
0.0399
0.2473
0.0276
0.0151
-0.0157
-0.2517
0.0112
0.1209
-0.0067
-0.5885
0.0102
0.1394
-0.0172
-0.5510
0.0255
0.0196
-0.0028
-1.0000
0.0073
0.2124
-0.0276
-0.7467
0.0558
0.0005
2DL5
0.0304
0.1782
0.0158
0.0662
0.0822
0.4808
0.1150
<0.0001
-0.0227
-0.3775
0.0229
0.0268
-0.0016
-0.1440
0.0006
0.7304
-0.0207
-0.6887
0.0361
0.0054
0.0019
1.0000
0.0033
0.3977
-0.0215
-0.6049
0.0332
0.0077
0.1311
0.5720
0.2969
<0.0001
2DS3
0.0779
0.4312
0.1017
<0.0001
0.1153
0.7789
0.2382
<0.0001
-0.0424
-0.6450
0.0848
<0.0001
0.0067
1.0000
0.0107
0.1301
0.0091
0.4947
0.0075
0.2066
0.0017
1.0000
0.0026
0.4524
-0.0109
-0.2791
0.0090
0.1661
0.0690
0.3159
0.0868
<0.0001
0.1074
0.5109
0.2060
<0.0001
2DS5
0.1190
0.7609
0.2501
<0.0001
0.0314
0.2517
0.0193
0.0421
-0.0108
-0.1513
0.0060
0.2566
-0.0037
-0.2819
0.0035
0.3889
-0.0171
-0.4777
0.0283
0.0138
0.0014
1.0000
0.0020
0.5079
-0.0330
-0.7790
0.0900
<0.0001
0.1284
0.6961
0.3271
<0.0001
0.1027
0.5786
0.2051
<0.0001
2DS1
0.0198
0.1504
0.0076
0.2027
0.0390
0.1973
0.0252
0.0202
-0.0113
-0.2113
0.0055
0.2774
-0.0003
-0.0360
0.0000
0.9398
-0.0173
-0.6495
0.0246
0.0217
0.0022
1.0000
0.0044
0.3344
-0.0315
-1.0000
0.0697
0.0001
0.1522
0.7475
0.3891
<0.0001
0.1223
0.5668
0.2465
<0.0001
2DS4
0.0316
0.1511
0.0163
0.0620
-0.0090
-0.3920
0.0064
0.2427
-0.0011
-0.1894
0.0002
0.8192
-0.0010
-1.0000
0.0011
0.6227
0.0158
0.3258
0.0968
<0.0001
-0.0003
-1.0000
0.0003
0.8070
0.0480
0.9113
0.7628
<0.0001
-0.0294
-0.8628
0.0684
0.0001
-0.0234
-0.7147
0.0426
0.0025
2DS3
2DS5
2DS1
aD,
D
D’
r2
P
D
D’
r2
P
D
D’
r2
P
-0.0065
-0.0593
0.0009
0.6673
0.0605
0.3240
0.0636
0.0002
0.1482
0.9407
0.4160
<0.0001
-0.0079
-0.2190
0.0051
0.2977
-0.0297
-0.7606
0.0788
<0.0001
-0.0291
-1.0000
0.0640
0.0002
The classical linkage disequilibrium coefficient measuring deviation from random association between alleles at different
loci [40]; D’, the linkage disequilibrium coefficient D standardized by the maximum value it can take (Dmax), given the allele
frequencies [41]; r2, another way to standardise the simple measure of linkage disequilibrium [36]; P, statistical significance.
24
Table 3. Pairs of KIR loci that displayed significant (p<0.05) LD in Macedonian
population.
KIR
locus
Positive LD
Negative LD
2DS2
2DL2, 2DS3, 2DS5, 3DP1
2DL3, 2DP1
2DL2
2DS2, 3DS1, 2DL2, 2DS3,
2DL3, 2DL1
2DS52DS1
2DL3
2DP1, 2DL1, 3DP1
2DS2, 2DL2, 2DL5, 2DS3
2DP1
2DL3, 2DP1, 3DP1
2DS2
2DL1
2DL3, 2DP1
2DL2
3DP1
2DL3, 2DP1
2DS2
3DL1
2DL1
3DS1, 2DL5, 2DS5, 2DS1, 2DS4
3DS1
2DL2,
2DS1
2DL5
2DL2, 3DS1, 2DS3, 2DS5,
2DL3, 2DL1, 3DL1, 2DS4
2DS1
2DS3
2DS2,
2DS1
2DL2,
3DS1,
2DL5,
2DS5
2DS2,
2DS1
2DL2,
2DS1,
2DL5,
2DS1
2DL2, 2DS1, 2DL5
2DL5,
2DS3,
2DS5,
2DL1, 3DL1, 2DS4
2DL3
2DL1, 3DL1, 2DS4
2DL1, 3DL1, 2DS4
25
Table 4. Diversity of investigated 33 populations for KIR genes locus by locus.
Locus
2DL1
2DL2
2DL3
2DL4
2DL5
3DL1
3DL2
3DL3
2DS1
2DS2
2DS3
2DS4
2DS5
3DS1
All loci
Gst
0.131
0.048
0.133
0.094
0.049
0.060
No diversity
No diversity
0.046
0.037
0.027
0.123
0.037
0.047
0.067
Ht
0.273
0.422
0.410
0.120
0.440
0.349
No diversity
No diversity
0.360
0.415
0.251
0.387
0.330
0.333
0.285
26
Hs
0.238
0.402
0.356
0.011
0.419
0.328
No diversity
No diversity
0.343
0.399
0.244
0.339
0.318
0.331
0.317
Table 5. Investigated populations, number of individuals, heterozygousity, and
standard genetic distances (SGD) between Macedonian population (present study)
and different populations (www.allelefrequencies.net) using KIR gene frequencies
(44).
Population
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
31.
32.
33.
France
Guadeloupe
Senegal
Finland
Reunion
Macedonia
Korea
Japan
Africa
SouthAsia
Basque
Mestizo
Huichol
Purepecha
Tarahumara
Argentina
Belgium
BrazilSouthEastCaucasian
EnglandWestMidlandsAfroCaribbean
EnglandWestMidlandsCaucasian
EnglandWestMidlandsIndianAsian
IndiaNorthHindus
IrelandNorthern
Italy
Lebanon
MexicoVeraCruzMestizos
PakistanKarachi
SpainGranada
SwedenVasterbotten
HongKongChinese
Oman
RomaniaCaucasians
SouthAfricaXhosa
Number of
individuals
108
118
90
102
101
214
154
41
62
108
71
86
73
103
75
102
100
55
54
126
35
72
100
217
120
51
78
100
150
100
99
123
50
27
Heterozygosity
± SE
0.270 ± 0.042
0.250 ± 0.050
0.179 ± 0.048
0.264 ± 0.048
0.307 ± 0.046
0.299 ± 0.045
0.193 ± 0.036
0.152 ± 0.035
0.249 ± 0.047
0.356 ± 0.048
0.302 ± 0.051
0.203 ± 0.046
0.222 ± 0.051
0.219 ± 0.055
0.216 ± 0.057
0.253 ± 0.040
0.250 ± 0.043
0.267 ± 0.049
0.260 ± 0.047
0.295 ± 0.045
0.323 ± 0.050
0.354 ± 0.053
0.270 ± 0.041
0.277 ± 0.044
0.282 ± 0.047
0.317 ± 0.051
0.374 ± 0.048
0.300 ± 0.047
0.297 ± 0.047
0.231 ± 0.037
0.275 ± 0.044
0.274 ± 0.048
0.232 ± 0.061
SGD
(x 102)
0.14
1.05
1.93
1.11
0.80
Reference
number
This paper
2.12
2.76
1.19
1.68
0.98
1.96
2.48
3.42
4.04
0.45
0.44
3.52
1.03
0.15
0.39
2.73
0.08
0.20
0.34
0.43
2.51
0.00
0.25
1.17
0.38
2.48
4.33
Figure legends
Figure 1: Killer cell immunoglobulin-like receptor (KIR) locus profiles observed (KIR
genotypes) and the number of individuals displaying each profile.
Figure 2: Neighbour-joining tree from genetic distances based on KIR gene
frequencies in Macedonian population and 32 previously reported populations ().
Grou
p
Genotype
ID
AA
AB
AB
AB
AB
AB
AB
AB
AB
AB
AB
AB
AB
AB
AB
AB
AB
AB
AB
AB
AB
AB
AB
AB
AB
AB
AB
AB
AB
AB
AB
AB
AB
AB
BB
1
2
3
4
5
6
7
8
9
10
11
13
14
15
16
18
19
23
28
33
62
63
200
202
205
233
260
268
319
336 new
370 new
371 new
372 new
373 new
69
3DL1
2DL1
2DL3
2DS4
2DL2
2DL5
3DS1
2DS1
2DS2
2DS3
28
2DS5
2DL4
3DL2
3DL3
2DP1
3DP1
Individuals
%
46
11
4
27
8
8
6
2
5
1
6
6
3
9
1
2
1
1
1
1
3
2
1
3
1
5
2
1
2
1
3
1
1
2
2
21,50
5,14
1,87
12,62
3,74
3,74
2,80
0,93
2,34
0,47
2,80
2,80
1,40
4,21
0,47
0,93
0,47
0,47
0,47
0,47
1,40
0,93
0,47
1,40
0,47
2,34
0,93
0,47
0,93
0,47
1,40
0,47
0,47
0,93
0,93
BB
BB
BB
BB
BB
BB
BB
BB
BB
BB
BB
BB
BB
BB
BB
BB
BB
BB
BB
BB
BB
2
4
1
5
2
1
2
1
1
1
3
1
1
1
1
1
1
3
1
1
1
70
71
72
73
76
87
90
113
159
294
317
318
359 new
363 new
374 new
375 new
376 new
377 new
378 new
379 new
380 new
Figure 1.
29
0,93
1,87
0,47
2,34
0,93
0,47
0,93
0,47
0,47
0,47
1,40
0,47
0,47
0,47
0,47
0,47
0,47
1,40
0,47
0,47
0,47
IndiaNorthHindus
PakistanKarachi
SouthAsians
EnglandWestMidlandsIndianAsian
BrazilSouthEastCaucasian
RomaniaCaucasians
MexicoVeraCruzMestizos
Basque
EnglandWestMidlandsCaucasian
Reunion
Macedonia
SpainGranada
IrelandNorthern
Africans
EnglandWestMidlandsAfroCaribbean
Italy
France
Argentina
Finland
HongKongChinese
Koreans
Japanese
Mestizo
Huichol
Purepecha
Tarahumara
SwedenVasterbotten
Oman
Lebanon
BelgiumKIRPop
Guadeloupe
Senegal
SouthAfricaXhosa
0.012
0.010
0.008
0.006
0.004
0.002
Figure 2.
30
0.000