insights on the hla-g evolutionary history provided by a nearby alu insertion

Article

Insights on the HLA-G Evolutionary History Provided by aNearby Alu InsertionKaisson E Santos1 Thalitta HA Lima1 Leandro P Felıcio1 Juliana D Massaro2 Gustavo M Palomino2

Ana Carolina A Silva3 Silviene F Oliveira3 Audrey Sabbagh45 Andre Garcia45 Philippe Moreau67

Eduardo A Donadi2 Celso T Mendes-Junior8 and Erick C Castelli9

1Instituto de Ciencias Biologicas Universidade Federal de Goias Goiania-GO Brasil2Divisao de Imunologia Clınica Departamento de Clınica Medica Faculdade de Medicina de Ribeirao Preto Universidade de SaoPaulo USP Ribeirao Preto SP Brasil3Laboratorio de Genetica Instituto de Ciencias Biologicas Universidade de Brasılia Brasılia-DF Brasil4Institut de Recherche pour le Developpement UMR 216 Mere et enfant face aux infections tropicales Universite Paris Descartes 4avenue de lrsquoObservatoire Paris France5Faculte de Pharmacie Universite Paris Descartes Sorbonne Paris Cite 4 avenue de lrsquoObservatoire Paris France6Commissariat a lrsquoEnergie Atomique et aux Energies Alternatives Institut des Maladies Emergentes et des Therapies InnovantesService de Recherches en Hemato-Immunologie Hopital Saint-Louis Paris France7Universite Paris-Diderot Sorbonne Paris-Cite UMR E5 Institut Universitaire drsquoHematologie Hopital Saint-Louis Paris France8Departamento de Quımica Faculdade de Filosofia Ciencias e Letras de Ribeirao Preto Universidade de Sao Paulo Ribeirao PretoSP Brasil9Departamento de Patologia Faculdade de Medicina de Botucatu Universidade Estadual Paulista ndash UNESP Botucatu SP Brasil

Corresponding author E-mail castellifmbunespbr

Associate editor Meredith Yeager

Abstract

The AluyHG element belongs to the AluYb8 subfamily It is a polymorphic insertion located approximately 20 kb fromthe HLA-G 30-untranslated region (30-UTR) which has been used for evolution studies because it exhibits identity fordescendants and it is still polymorphic in the human genome To understand the evolutionary mechanisms acting onHLA-G we evaluated the presence or absence of the AluyHG element associating this variable site with others observedat HLA-G coding 30-UTR or both regions in four distinct populations (Brazilian French Congolese and Senegalese)The results were compared with the 1000Genomes Consortium data The worldwide AluyHG frequencies showed anincrement starting lower in Africa and increasing following distance and time of human dispersion out of Africa Thesame haplotype pattern was observed in all populations indicating that most of the HLA-G haplotypes already detectedwere originated earlier in Africa before Homo sapiens dispersion The AluyHG insertion was associated with theG01010101UTR-1 haplotype with rare recombinants Despite its high frequency in worldwide populations theG01010101UTR-1 haplotype should be very recent The low frequency of recombinants indicates that the rate ofrecombination at the HLA-G gene is very low

Key words AluyHG HLA-G 30-untranslated region haplotypes worldwide diversity human evolution

IntroductionThe human major histocompatibility complex (MHC) com-prises at least 224 genes at chromosome 6p213 coding thehuman leukocyte antigens (HLAs) that have a key role on theimmune system Classical class I genes (HLA-A HLA-B andHLA-C) encode molecules that present antigen peptides to TCD8 + cells whereas the nonclassical class I genes (HLA-GHLA-E and HLA-F) have been primarily associated with themodulation of the immune system cells (Klein and Sato 2000Hviid 2006 Donadi et al 2011)

HLA-G has been considered to be an immune modulatorymolecule predominantly expressed at the maternalndashfetalinterface and has primarily been associated with maternalndashfetal tolerance (Hviid 2006 Carosella et al 2008 Berger et al2010) The complete HLA-G molecule presents the same

extracellular structure of the classical HLA counterparts how-ever its major function is not antigen presentation HLA-Ginhibits the cytotoxic activity of T CD8 + and NK cellsthrough direct interaction with leukocyte receptors such asILT-2 (LILRB1 and CD158j) ILT-4 (LILRB2 and CD58d) andKIR2DL4 (CD158d) (Colonna 1997 Ponte et al 1999Rajagopalan and Long 1999 Gao et al 2000 Contini et al2003 Shiroishi et al 2006 Donadi et al 2011) HLA-G gene andmolecule expression patterns differ in relation to classical HLAclass I molecules in many aspects including 1) restrictedtissue expression in nonpathological conditions (Lee et al1995) being expressed at the maternalndashfetal interface inthe extravillous cytotrophoblast cells (Berger et al 2010)thymus cornea proximal nail matrix pancreas and hemato-poietic stem cells (Crisa et al 1997 Mallet et al 1999

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Mol Biol Evol 30(11)2423ndash2434 doi101093molbevmst142 Advance Access publication August 14 2013 2423

Le Discorde et al 2003 Menier et al 2004 Ito et al 2005 Cirulliet al 2006) 2) the presence of several membrane and solubleisoforms due to alternative splicing of the full-length HLA-GmRNA (reviewed at [Hviid 2006 Donadi et al 2011]) 3) aunique short cytoplasmic tail due to the presence of a pre-mature stop codon at exon 6 (Hviid 2006 Donadi et al 2011)4) limited protein variability (Hviid 2006 Larsen and Hviid2009 Donadi et al 2011) 5) a unique 50URR (50 upstreamregulatory region) (Solier et al 2001 Moreau et al 2009) and6) a very polymorphic 50 promoter (Hviid et al 1999 2004Tan et al 2005 Hviid et al 2006 Castelli et al 2011 Martinez-Laso et al 2013) and 30-untranslated region (30-UTR) (Castelliet al 2010)

In pathological conditions HLA-G expression has beendetected in tumors chronic viral infections inflammatoryand autoimmune diseases and in engrafted tissues(LeMaoult et al 2003 Carosella et al 2008 Crispim et al2008 Amiot et al 2011 Silva et al 2011) In this contextthe expression of HLA-G in chronic inflammation autoim-mune diseases and allotransplants has been associated with abetter prognosis due to the inhibition of the immuneresponse In contrast the inhibition of the immune responsemay be harmful in chronic viral infections and tumorssituations in which a vigorous immune response would berequired (Carosella 2011)

In contrast to the classical HLA class I genes limited var-iability at the HLA-G coding region has been observed inworldwide populations (reviewed by Donadi et al [2011]and Larsen and Hviid [2009]) According to theInternational ImmunoGeneTics Database (IMGTDatabasedatabase version 3120 April 2013) 50 HLA-G alleles (or dif-ferent coding haplotypes) are currently described generating16 different full-length proteins Of these five alleles are fre-quently observed in worldwide populations and they wouldbe considered as polymorphisms (Castelli et al 2007 2011Donadi et al 2011) The low variability at the coding region isprobably associated with the very limited repertoire of pep-tides that HLA-G would present to T cells and also to ensurea nonvariable molecule structure permitting the modulationof the immune response in vital situations such as pregnancyIndeed it has been considered that purifying selection hasshaped the HLA-G coding region variability avoiding a highdegree of variation (Castro et al 2000) In contrast a relativelyhigher degree of variation has been observed at the HLA-G50URR (Hviid et al 1999 Tan et al 2005 Hviid et al 2006Castelli et al 2011 Martinez-Laso et al 2013) and 30-UTR(Castelli et al 2010) which may be highly implicated inHLA-G expression regulation (Hviid et al 2006 Donadi et al2011) mRNA stability (Hviid et al 2003 Rousseau et al 2003)mRNA degradation (Tan et al 2007 Yie et al 2008) andpossible differential activity of microRNAs (miRNA) (Tanet al 2007 Castelli et al 2009 Manaster et al 2012)

The HLA-G variability in the regulatory coding and 30UTregion has already been evaluated in a Brazilian admixedpopulation (Castelli et al 2011) This study together withothers that evaluated the promoter region indicated thatthe HLA-G gene presents a high linkage disequilibrium (LD)along the entire gene exhibiting few extended haplotypes and

a haplotype pattern that seems to be the same worldwide(Ober and Aldrich 1997 Hviid et al 1999 2004 Tan et al 2005Hviid et al 2006 Larsen and Hviid 2009 Cervera et al 2010Castelli et al 2011 Donadi et al 2011 Lucena-Silva et al 2012Courtin et al 2013 Di Cristofaro et al 2013 Garcia et al 2013Martinez-Laso et al 2013 Sabbagh et al 2013)

In humans Alu sequences are the most abundantelements of the SINE (Short Interspersed Nuclear Element)retrotransposon family presenting approximately 300 bp(Rowold and Herrera 2000) Alu retroelements have beenamplified within the human genome during recent evolution-ary period and are useful polymorphic markers for humanpopulation origin studies (Batzer and Deininger 2002 Kulskiand Dunn 2005) These elements have been classified intodifferent subfamilies (AluJ AluS and AluY) based on theirmutation pattern genetic age and sequence difference(Jurka and Smith 1988 Batzer and Deininger 2002) Amongthe AluY subfamilies the AluYb8 is a human-specific poly-morphic element that has been used as a relevant toolfor population studies Five different AluYb8 sequenceshave already been identified in the human MHC class Iregion ie AluyMICB AluyTF AluyHJ AluyHF and AluyHG(Kulski and Dunn 2005)

Among the features that turn the AluyHG element into aninteresting marker for population and HLA-G studies are 1) itbelongs to the AluYb8 subfamily and is located approximately20 kb downstream from the 30-UTR of the HLA-G gene (Kulskiet al 2001) 2) this mobile element is dimorphic ie is eitherpresent or absent (Rowold and Herrera 2000) 3) it exhibitsidentity for descendants ie if two individuals share an inser-tion at the same locus they have a common ancestor (Rowoldand Herrera 2000) 4) this marker is not fixed in the humangenome ie it is still polymorphic (Kulski and Dunn 2005)5) its ancestral state is known ie the element absence isthe original state (Rowold and Herrera 2000) and 6) theAluyHG element evaluation is straightforward and simpleFurthermore the AluYb8 subfamily seems to be transposi-tionally active and seems to be a human-specific fragment(Zietkiewicz et al 1994) The AluYb8 retrotranspositionmechanism involves the transposition of a RNA polymeraseIII retrotranscribed product probably by a reverse transcrip-tase encoded into a LINE sequence (Long InterspersedNuclear Element) because the Alu elements do not presentencoded reverse transcriptase (Batzer and Deininger 2002)

To better understand the evolutionary mechanisms actingon the HLA-G gene the presence or absence of the AluyHGelement was evaluated associating this Alu site with othervariation sites at the coding 30-UTR or both regions in fourdistinct populations (Brazilian French Congolese andSenegalese) To evaluate whether the pattern observed byus was also present worldwide we compared our resultswith those available at the 1000Genomes Consortium data(1000Genomes Project Consortium 2012)

ResultsThe presence or absence of the AluyHG element was evalu-ated in 641 individuals 165 Brazilians (BRA) 161 Congolese(CNG) 193 Senegalese (SEE) and 122 French (FRE) The

2424

Santos et al doi101093molbevmst142 MBE

frequencies of the absence (AluyHG1) or presence(AluyHG2) are presented in table 1 All genotype frequenciesdid fit the HardyndashWeinberg expectations for all populationsIn addition table 1 presents data regarding the AluyHG fromdifferent populations studied so far The frequency of theAluyHG2 varies among different populations Populationsfrom East Asia including Malaysian Chinese Hunan HanInner Mongolian Han Inner Mongolian Mongol andGuangdong Han usually present higher AluyHG2 frequen-cies compared with any other population studied so far whilepopulations from Africa including Congolese SenegaleseSoutheastern Bantus Khoi Sekele San and Kung San usuallypresent lower frequencies of this Alu element The Brazilianand French populations evaluated in the present studyexhibited intermediate frequencies of the AluyHG element

The presence of association between AluyHG and HLA-G30-UTR variable sites was evaluated by Haploview as de-scribed in the Materials and Methods section Given the pos-itive association but unknown gametic phase haplotypeswere inferred by probabilistic models The 30-UTR haplotypesfound in Brazil France and Africa were named accordingto previous studies (Castelli et al 2010 Lucena-Silva et al2012) and data are presented in table 2 We noticed thatthe presence of AluyHG was mainly associated with the pres-ence of the UTR-1 haplotype In fact the only exception wastwo individuals from France (082 of the French samples)with the UTR-3AluyHG2 haplotype Thus the frequency ofAluyHG2 was compatible with the UTR-1 frequencies Thishaplotype is the most frequent in Brazil and the second mostcommon in France whereas its frequency is low in Africa

Besides the association between UTR-1 and AluyHG pre-sented above haplotypes UTR-1AluyHG1 were detected

(table 2) in all studied populations The higher frequency ofthis haplotype was detected in France Nevertheless the fre-quency of this haplotype was quite low in which most ofthe UTR-1 found in any of the four populations studied

Table 1 Frequency of AluyHG Absence (AluyHG1) or Presence (AluyHG2) in Worldwide Populations

Continent Population N AluyHG1 AluyHG2 Reference

Africa

Sekele San 60 09670 00330 Kulski and Dunn (2005)Southeastern Bantus 50 09400 00600 Kulski and Dunn (2005)Kung San 42 09270 00730 Kulski and Dunn (2005)Khoi 43 08690 01310 Kulski and Dunn (2005)Senegalese 193 08964 01036 Current studyCongolese 161 08944 01056 Current study

Asia

Northeastern Thai 192 07080 02920 Dunn et al (2006)Japanese 99 07300 02700 Kulski and Dunn (2005)Mongolians 41 07800 02200 Kulski and Dunn (2005)Malaysian Chinese from Malaysia 50 04400 05600 Dunn et al (2007)Hunan Han 147 05646 04354 Tian et al (2008)Inner Mongolian Han 104 06106 03894 Tian et al (2008)Inner Mongolian Mongol 87 06322 03678 Tian et al (2008)Guangdong Han 107 06121 03879 Tian et al (2008)

Oceania Australian Europeans 105 06990 03010 Kulski and Dunn (2005)

Europe French 122 07787 02213 Current study

America

Brazilian Cohort 1a 165 07333 02667 Current studyBrazilian Cohort 2b 101 07570 02430 Silva ACAd

Brazilian Cohort 3c 61 07000 03000 Silva ACAd

NOTEmdashN = number of individualsaBrazilians from Ribeirao Preto Sao Paulo BrazilbBrazilians from Brasılia-DF BrazilcKalunga (Afro-derived Brazilian population)dSilva ACA et al (personal communication)

Table 2 HLA-G 30-UTRAluyHG Haplotypes in the Four PopulationsEvaluatedmdashBrazilian Congolese Senegalese and French

HLA-G30-UTRhaplotypec

AluyHGallele

Frequencies

Braziliansa Congolese Senegalese French

N 152b 161 193 122

UTR-1 Present 02500 01056 01036 02131

UTR-1 Absent 00230 00342 00052 00533

UTR-2 Absent 02500 02112 03575 02664

UTR-3 Present mdash mdash mdash 00082

UTR-3 Absent 01250 03043 02798 01352

UTR-4 Absent 01282 01056 00518 01434

UTR-5 Absent 00757 00932 01321 00369

UTR-6 Absent 01020 01304 00648 00861

UTR-7 Absent 00428 mdash mdash 00533

UTR-8 Absent 00033 00031 mdash mdash

UTR-15 Absent mdash 00124 mdash 00041

UTR-16 Absent mdash mdash 00052 mdash

UTR-17d Absent mdash 00093 mdash mdash

UTR-13 Absent mdash 00031 mdash mdash

NOTEmdashN = number of individualsaBrazilians from Ribeirao Preto Sao Paulo BrazilbThis sample size differs from the original because HLA-G 30-UTR variability was notavailable for 13 samplescHaplotypes were named according to Castelli et al (2010) and Lucena-Silva et al(2012)dThis 30-UTR haplotype was not detected in the studies by Castelli et al (2010) andLucena-Silva et al (2012)

2425

Insights on HLA-G Evolutionary History doi101093molbevmst142 MBE

were associated with the presence of the AluyHG element(allele AluyHG2)

To evaluate the HLA-G haplotype pattern found world-wide and compare the results with data shown in table 2 weevaluated LD between variable sites within or close to theHLA-G gene obtained from the 14 populations evaluated bythe 1000Genomes Project as described in the Materials andMethods section For that purpose variable sites from theentire coding and 30-UTR were used (fig 1) with each variablesite identified by its position following the Adenine of the firsttranslated ATG as nucleotide + 1 Because the same set ofBrazilian samples used in this article was already evaluatedfor the HLA-G coding and 30-UTR variability (Castelli et al2011) we opt to concatenate data from Brazil with the1000Genomes data (fig 1) However as the AluyHG elementis not included in the 1000Genomes data we also evaluatedthe LD among the HLA-G 30-UTR variable sites and theAluyHG element in Brazilian Congolese Senegalese andFrench populations (fig 2)

The LD evaluation in worldwide populations indicatedthe presence of significant LD along the entire HLA-G geneusually presenting a single segregation block encompassingthe coding 30-UTR and surrounding variable sites (fig 1) Inaddition the same 30-UTR variable sites were also in LD withthe AluyHG element as shown in figure 2 Considering theLD pattern found in figure 1 haplotypes were inferred asdescribed in the Materials and Methods section Table 3

FIG 1 LD across the HLA-G gene including variable sites at the coding region and 30-UTR by using data from the 1000Genomes Consortium and of theBrazilian population Areas in dark red or dark gray indicate strong LD (LOD 2 D0 = 1) shades of pink or shades of gray indicate moderate LD(LOD 2 D0lt 1) blue or light gray indicates weak LD (LODlt 2 D0 = 1) and white indicates no LD (LODlt 2 D0lt 1) D0 values different from 100 arerepresented inside the squares as percentages LOD log of the odds D0 pairwise correlation between single-nucleotide polymorphisms

FIG 2 LD across the HLA-G 30-UTR and the AluyHG element byusing data from Brazilian Senegalese Congolese and French popula-tions Areas in dark red or dark gray indicate strong LD (LOD 2D0 = 1) shades of pink or shades of gray indicate moderate LD(LOD 2 D0lt 1) blue or light gray indicates weak LD (LODlt 2D0 = 1) and white indicates no LD (LODlt 2 D0lt 1) D0 values dif-ferent from 100 are represented inside the squares as percentagesLOD log of the odds D0 pairwise correlation between single-nucle-otide polymorphisms

2426


Tab

le3

Freq

uen

cyof

the

HLA

-GC

odin

g30

-UT

RH

aplo

typ

esin

Wor

ldw

ide

Popu

lati

on

Euro

pe

Asi

aA

fric

aA

mer

ica

Pop

ula

tion

British(EnglandScotland THORN

Finnish(Finland)

Iberian(Spain)

Tuscany(Italy)

HanChinese(SouthChina)

HanChinese(IBeijingChina)

Japanese

(TokyoJapan)

Yoruba(IbadanNigeria)

Luhya

(WebuyeKenya)

PuertoRican(PuertoRico)

Colombian(Medellin

Colombia)

MexicanAncestry

(LosAngelisUSA)

EuropeanAncestry

(UtahUSA)

AfricanAncestry

(SouthwestUSA)

Braziliansa

(SoutheasternBrazil)

HLA

-G30

-UT

RH

aplo

typ

esb

HLA

-GC

odin

gH

aplo

typ

esc

N89

9314

9810

097

8988

9755

6066

8561

108

UT

R-1

G0

101

01

010

3371

036

020

3214

029

080

4250

029

470

2619

012

180

2391

027

780

2373

027

270

3941

021

820

2360

G0

101

01

04mdash

mdashmdash

mdashmdash

mdashmdash

002

560

0109

000

93mdash

000

76mdash

000

91mdash

G0

101

03

G0

101

01

01d

mdashmdash

mdashmdash

000

50mdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdash

UT

R-2

G0

101

02

010

2303

017

740

3571

020

410

0400

011

050

1607

014

100

1848

011

110

1780

021

270

2059

020

910

1530

G0

101

02

02mdash

mdashmdash

000

51mdash

mdashmdash

000

640

0380

mdashmdash

mdashmdash

mdashmdash

G0

106

006

180

0269

003

570

0663

001

000

0263

000

60mdash

000

540

0278

004

240

0227

004

710

0091

005

10G

01

05N

mdash0

0108

mdash0

0255

001

500

0421

000

600

0962

005

43mdash

000

850

0303

000

590

0636

004

20G

01

05N

(+18

8C

)emdash

mdashmdash

001

53mdash

000

53mdash

001

280

0217

mdash0

0085

001

52mdash

001

82mdash

G0

101

03

01G

01

010

201

d0

0056

mdashmdash

mdashmdash

mdash0

0060

mdashmdash

mdashmdash

mdashmdash

001

82mdash

G0

101

14

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

000

50

UT

R-3

G0

104

01

004

490

0591

007

140

0969

025

500

2474

045

240

0256

001

090

1296

017

800

1515

006

470

0364

004

60G

01

040

3mdash

mdashmdash

mdash0

0050

000

530

0179

mdashmdash

mdashmdash

mdashmdash

mdash0

0050

G0

104

04

001

120

0054

mdash0

0306

mdashmdash

mdash0

2244

009

780

0556

003

390

0076

002

350

1000

003

20G

01

040

5mdash

mdashmdash

mdashmdash

mdashmdash

000

640

0109

000

930

0169

mdashmdash

000

91mdash

G0

101

08

mdashmdash

mdashmdash

mdash0

0053

001

19mdash

mdashmdash

mdashmdash

mdashmdash

mdashG

01

040

4(+

188

C)e

mdashmdash

mdashmdash

mdashmdash

mdash0

0064

mdashmdash

mdashmdash

mdashmdash

mdashG

01

010

3G

01

040

1dmdash

mdashmdash

mdash0

0050

001

580

0119

mdashmdash

mdashmdash

mdashmdash

mdashmdash

UT

R-4

G0

101

01

050

1180

028

490

1071

014

290

0200

004

740

0060

011

540

0761

012

960

1356

007

580

1529

005

450

0970

G0

101

09

mdashmdash

mdashmdash

mdashmdash

mdash0

0321

002

72mdash

000

850

0076

mdash0

0091

000

50

UT

R-5

G0

103

002

810

0161

mdash0

0306

mdash0

0263

001

790

0897

008

700

1111

007

630

0985

003

530

1364

008

80G

01

040

10

0056

mdashmdash

000

51mdash

mdashmdash

mdashmdash

000

93mdash

mdashmdash

mdashmdash

G0

101

08

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

000

90

(con

tin

ued)

2427


Tab

le3

Con

tin

ued

Euro

pe

Asi

aA

fric

aA

mer

ica

Pop

ula

tion


Finnish(Finland)

Iberian(Spain)

Tuscany(Italy)



Japanese

(TokyoJapan)


Luhya

(WebuyeKenya)


Colombian(Medellin

Colombia)

MexicanAncestry

(LosAngelisUSA)

EuropeanAncestry

(UtahUSA)

AfricanAncestry

(SouthwestUSA)

Braziliansa


HLA

-G30

-UT

RH

aplo

typ

esb

HLA

-GC

odin

gH

aplo

typ

esc

N89

9314

9810

097

8988

9755

6066

8561

108

UT

R-6

G0

101

01

040

0618

001

080

0714

002

04mdash

mdashmdash

008

330

1359

008

330

0678

003

790

0118

010

000

0740

G0

101

01

01mdash

mdashmdash

000

51mdash

mdashmdash

mdashmdash

mdashmdash

mdash0

0059

mdash0

0090

G0

101

01

05mdash

mdashmdash

000

51mdash

mdashmdash

001

28mdash

mdashmdash

mdashmdash

mdash0

0149

G0

101

01

04(+

1019

C)d

mdashmdash

mdash0

0051

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdash

UT

R-7

G0

101

03

010

0899

004

840

0357

004

590

2200

016

840

0471

mdashmdash

004

630

0085

004

550

0471

000

910

0370

G0

101

05

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

000

50

UT

R-8

G0

106

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

001

40

UT

R-9

G0

101

08

000

56mdash

mdash0

0051

mdash0

0053

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdash

UT

R-1

8fG

01

010

201

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdash0

0076

mdashmdash

mdash

UT

R-1

9fG

01

010

301

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

000

59mdash

mdash

NO

TEmdash

N=

num

ber

ofin

divi

dual

sa Br

azili

ans

from

Rib

eira

oPr

eto

Stat

eof

Sao

Paul

oBr

azil

The

HLA

-Gva

riabi

lity

was

prev

ious

pub

lishe

dby

Cas

telli

etal

(2

011)

bH

LA-G

30-U

TR

hapl

otyp

esw

ere

nam

edac

cord

ing

toC

aste

lliet

al

(201

0)an

dLu

cena

-Silv

aet

al

(201

2)

c HLA

-Gco

din

gha

plo

typ

esw

ere

nam

edac

cord

ing

toth

eIn

tern

atio

nal

Imm

unog

enet

ics

Dat

abas

e(I

MG

TH

LA)

dR

ecom

bina

ntha

plot

ypes

e Li

kely

ance

stor

alle

lefo

llow

edby

the

mut

atio

nth

atde

fined

this

hapl

otyp

ef T

his

30-U

TR

hap

loty

pes

was

not

dete

cted

inth

est

udie

sby

Cas

telli

etal

(2

010)

and

Luce

na-

Silv

aet

al

(201

2)

2428


presents the 32 HLA-G coding30-UTR haplotypes found byusing the Brazilian (data already published by Castelli et al[2011]) and the 1000Genomes data The coding haplotypeswere named according to the known sequences described inthe IMGT database whereas 30-UTR were named accordingto previous studies (Castelli et al 2010 Lucena-Silva et al2012) In cases in which the haplotype was not compati-ble with an IMGT HLA-G allele the likely ancestor allelefollowed by the mutation that defined this new haplotypewas indicated In addition some possible recombinationevents between known HLA-G alleles were found and wereindicated

The association between the HLA-G coding30-UTR regionswas evaluated in worldwide populations (table 3) It can benoticed that the same pattern of coding and 30-UTR haplo-types found in Brazil (Castelli et al 2010 2011) was also foundin any population studied by the 1000Genomes ProjectConsortium (2012) and others (Hviid et al 2004 2006Larsen and Hviid 2009 Jassem et al 2012 Martinez-Lasoet al 2013) UTR-1 was found in all populations and it wasmainly associated with the coding allele G01010101 orwith recombinant haplotypes in which the last part ofthe sequence resembles the G01010101 allele Only a fewG01010104UTR-1 haplotypes were found mainly inAfrica (table 3) In Brazil all UTR-1 haplotypes were foundassociated with the allele G01010101 The G01010101UTR-1 haplotype frequencies ranged between 1218 forYoruba and 4250 for Han Chinese South population(table 3) Given the fact that the same Brazilian HLA-Gcoding30-UTR LD pattern was observed worldwide andthat the LD plot (fig 2) indicates that the LD extendsbeyond the AluyHG site the same 30-UTRAluyHG patternobserved in Brazilian Senegalese Congolese and French pop-ulations may be extrapolated to worldwide populations It isworthy mentioning that the presence of the AluyHG elementwas previously associated with HLA-A2 allele and also thatthis Alu element lays between the HLA-G and HLA-A lociindicating that the pattern of LD observed for HLA-G mightextends up to the HLA-A gene (Kulski et al 2001) In additionthe frequencies for the G01010101UTR-1 haplotype fol-lowed the same pattern observed for the AluyHG ie higherfrequencies in Asian populations and lower frequencies inAfrican populations (tables 2 and 3)

DiscussionThe present study is the first to evaluate the association be-tween the AluyHG element and variable sites at the HLA-Ggene We characterized 641 individuals from differentcountries including the Brazilian French Congolese andSenegalese populations for the presence or absence of theAluyHG element and evaluated the relationship betweenthis insertion and variable sites at the HLA-G coding regionand 30-UTR Moreover we compared our results with the1000Genomes data available in public databases andevaluated the HLA-G distribution pattern in worldwidepopulations

Previous studies (Dunn et al 2007 Tian et al 2008) alsonoticed that the AluyHG presence frequencies were higher in

East Asian populations mainly in Chinese (table 1) TheAluyHG frequencies in Brazil are quite similar to thoseobserved in Europe Japan and Thailand (table 1) The pop-ulation from the State of Sao Paulo which is included in thepresent manuscript (table 1) did present a major Europeancontribution in its gene pool (Ferreira et al 2006 Muniz et al2008) In addition the Brazilian frequencies observed in thepresent study were similar to those reported for the popula-tion of Brasilia (Brazilrsquos capital distant 706 km from RibeiraoPreto SP) and for the Kalunga population an Afro-derivedpopulation from the State of Goias Brazil (Silva ACA personalcommunication)

The insertion of an Alu element is considered to be arandom process occurring in any chromosome locationAlthough random this phenomenon might be influencedby specific target sequences (Jurka 1997) and the frequencyof specific haplotypes in a given chromosomal region Thusone may consider that it is possible that an insertion eventwould occur in certain haplotypes that are more frequentthan others The presence of the AluyHG element in all pop-ulations that were evaluated (table 1) led us to infer that thisinsertion event occurred in Africa probably before Homo sa-piens dispersion to other continents This proposal is based onthe fact that the insertion event is not reversible ie it was notdescribed a mechanism in which an Alu element is perfectlyremoved (Kulski et al 2001) In addition considering the lowfrequency of this element in any African population evalu-ated it is probable that this Alu insertion is a recent eventotherwise a greater frequency of this element might be ex-pected in such populations On the other hand despite suchlower African frequencies the insertion was observed in allAfrican populations studied so far (table 1) suggesting thatthe insertion may be old enough in order to spread acrossAfrica

The major founder event experienced by modern humanswhen leaving Africa (Henn et al 2012) may be responsible fora sharp increase in the frequency of the AluyHG element innon-African populations When comparing the frequencies ofthe AluyHG presence with the dispersion event and migratoryroutes followed by modern humans (Henn et al 2012) weobserved that there is a gradual frequency increment follow-ing space and temporal dispersion distances from Africa toother continents Thus the AluyHG increased frequenciesin populations outside Africa might be a consequence ofisolation by distance and selective pressures acting in thosefrequencies

Although neutral evolution may explain the AluyHG dis-tribution the bearing chromosomal region is one of the majortargets of natural selection in the human genome (Solberget al 2008) In fact evidences of balancing selection acting inthe regulatory regions of the HLA-G gene have been described(Tan et al 2005 Castelli et al 2011) Moreover the signatureof balancing selection acting on HLA-G may be due to bal-ancing selection acting in other genes in the same chromo-somal region (Gaudieri et al 2000) particularly the HLA-Agene because a strong LD was also described between theAluyHG element and the allele group HLA-A2 (Kulski et al2001) To elucidate some evolutionary mechanisms that

2429


acted on HLA-G we evaluated the LD pattern between theAluyHG element and variable sites at the HLA-G 30-UTRwhich is of great importance for the HLA-G post-transcriptionregulation and where balancing selection appears tohave maintained high heterozygosity (Castelli et al 2011Martinez-Laso et al 2013) Because this Alu insertion is anancient event that probably took place before the humandispersion out of Africa as discussed earlier and taking intoaccount its distance from the HLA-G gene (approximately20 kb) it is expected that many HLA-G 30-UTR haplotypeswould be associated with the AluyHG presence due to re-combination events However the AluyHG insertion only pre-sented a strong association with the UTR-1 haplotype despitethe occurrence of a rare recombinant haplotype (table 2) andwith the allele G01010101 as illustrated by the Braziliandata

The UTR-1 haplotype was theoretically considered as ahigh HLA-G producer haplotype presenting high frequenciesin worldwide populations (Castelli et al 2010 Donadi et al2011) This 30-UTR haplotype is associated with the codingallele G01010101 in all populations evaluated (table 3) andit was associated with higher soluble HLA-G production(Martelli-Palomino et al 2013) In addition UTR-1 does notpresent the 14 bp fragment which in turn was also associatedwith increased soluble HLA-G levels (Hviid et al 2004 Rizzoet al 2012 Svendsen et al 2013) Previous studies (Castro et al2000 Donadi et al 2011) together with the present dataindicated that the G01010101UTR-1 haplotype wouldbe the most recent one among the frequent extendedhaplotypes described for HLA-G Although this informationis not compatible with the high frequency of this haplotypeobserved in all populations evaluated it is in agreement withthe low frequencies of the haplotype in Africa (table 3)Despite being recent balancing selection may have essentiallyshaped the G01010101UTR-1 frequencies all over theworld

This G01010101UTR-1 haplotype would be associatedwith high HLA-G production by a combination of featuresthat include more stable mRNAs (Yie et al 2008) low affinityof microRNAs (Tan et al 2007 Castelli et al 2009 Manasteret al 2012) and a unique 50 regulatory region (Hviid et al1999 Solier et al 2001 Tan et al 2005 Castelli et al 2011Martinez-Laso et al 2013) It is reasonable to propose thatnatural selection shaped the frequency of this haplotype lead-ing to high frequencies of G01010101UTR-1 all over theworld (table 3) but also high heterozygosity (compatible withbalancing selection as previously observed for the HLA-G reg-ulatory regions) Since the differential expression of HLA-Gmay be beneficial or harmful depending on the underlyingcondition HLA-G heterozygosis would prone the individual toface different situations

Nevertheless the fact that practically no recombinantswere found (99 of the AluyHG were associated with UTR-1 considering Brazil Congo Senegal and France and 100 ofthe AluyHG were associated with G01010101UTR-1 con-sidering only Brazil) and the presence of low frequencies ofAluyHG in Africa we may postulate that G01010101UTR-1is in fact the most recent haplotype among the frequent ones

Otherwise a great recombination rate would be found as wellas other haplotypes either ancestral or derived from theG01010101UTR-1 haplotype would also present withthis AluyHG In fact it is possible that the AluyHG elementinsertion might have occurred earlier in the emergence of theG01010101UTR-1 haplotype (figs 3 and 4)

The AluyHG frequencies were compatible to the UTR-1frequencies (tables 1 and 2) in the populations evaluated inthe present manuscript probably due to a hitchhiking effectIn addition a low recombination rate was observed for theHLA-G gene and surrounding variable sites with a haplotypeblock extending beyond 20 kb from HLA-G 30-UTR encom-passing the AluyHG site (figs 1 and 2) and possible up to theHLA-A gene (Kulski et al 2001) Therefore the evolutionaryevents that shaped the HLA-G coding and 30-UTR frequenciesall over the world also shaped the AluyHG frequencies Thisconserved haplotype block theoretically might be due to theimportant role of HLA-G in the modulation of immune re-sponses and immune tolerance (Hviid 2006 Larsen and Hviid2009 Donadi et al 2011)

The higher G01010101UTR-1 haplotype frequency wasfound in the Chinese Han population which is in accordancewith the high frequency of the AluyHG element in EasternAsia (Dunn et al 2007 Tian et al 2008) Interestingly thesepopulations do not present the UTR-6 haplotype (table 3) orits associated coding allele G01010105 It is possible thatUTR-6 may have been lost in this population by the occur-rence of either genetic drift or a different selective pressure Incontrast African and Asian populations present higher UTR-3frequency (table 3) which was also observed in the samplesfrom Congo and Senegal (table 2) However the UTR-3 fre-quencies decreased from Africa to the other continents prob-ably due to founder effects coupled with selective pressuresThe African population including our samples from Congoand Senegal missed the UTR-7 haplotype which was recentlyassociated with lower soluble HLA-G levels (Martelli-Palomino et al 2013) However further studies are necessaryto elucidate this scenario

In conclusion our data support the evidence that theG01010101UTR-1 haplotype would be one of the mostrecent haplotypes although originated before the dispersionout of Africa The G01010101UTR-1AluyHG frequenciesall over the world (and also for other HLA-G haplotypes)might be a consequence of the sum of consecutive foundereffects as well as selection modulating its frequency

Materials and MethodsFour distinct populationsmdashhealthy unrelated individuals ran-domly selected from Brazil Congo Senegal and Francemdashwere evaluated regarding the HLA-G variability and theAluyHG element The Brazilian population was composedof 165 individuals from Ribeirao Preto State of Sao PauloBrazil the Congolese population comprised 161 individualsfrom the Badundu province of the Democratic Republic ofthe Congo Senegalese population comprised 193 individualsfrom the Niakhar area Senegal and the French populationcomprised 122 individuals from Paris France The LocalResearch Ethics Committee approved the protocol of the

2430


study for Brazil and Senegal The Ministry of Congo and theFrench Government approved this protocol The HLA-Gcoding region and 30-UTR variability in Brazilians was alreadyreported in a previous study (Castelli et al 2010 2011) The30-UTR variability of the African and French samples was re-ported in recent studies (Courtin et al 2013 Garcia et al 2013Martelli-Palomino et al 2013 Sabbagh et al 2013)

The AluyHG element was evaluated by using a previouslypublished procedure (Kulski et al 2001) Briefly the DNA wasamplified by polymerase chain reaction (PCR) using the

primers AluyHGF-CAGGACAACCAGTAAAGATGCTGG andAluyHGR-GCTTCAGTTAACATGCAAGTTTATGCC The reac-tion was carried out in 25mL containing 20 ng of templateDNA 15 pmol of each primer (AccuOligo Bioneer Korea)02 mM dNTPs (Fermentas Vilnius Lithuania) 10 unit of TaqPlatinum Polymerase (Invitrogen Carlsbad CA USA) 08 ofPCR buffer and 5 DMSO The cycling conditions comprisedan initial denaturation at 94 C for 3 min followed by 30cycles at 94 C for 30 s 58 C for 30 s 72 C for 50 s andfinal extension at 72 C for 5 min The amplification was

FIG 3 Network from HLA-G coding region and 30-UTR haplotypes considering worldwide population (data from the 1000Genomes Consortium) Thenetwork was calculated by Median-Joining method (Bandelt et al 1999) using the Network Program version 4611 considering the 1000Genomes dataand excluding the haplotypes with frequencies lower than 1 The coding haplotypes were named according to the known sequences described in theIMGT database whereas 30-UTR were named according to earlier studies (Castelli et al 2010 Lucena-Silva et al 2012) The HLA-G Lineages were namedaccording to Castelli et al (2011) and different colors indicate different HLA-G lineages The black arrow indicates when the AluyHG took place Theblack boxes indicate the number of mutational steps between different haplotypes where 7 indicates the + 15 + 36 + 147 + 188 + 372 + 1019 and+ 3142 mutations 6 indicates the + 15 + 36 + 292 + 372 + 1054 and + 1590 mutations 2a indicates + 706 and + 3196 mutations 2b indicates+ 748 and + 3027 mutations 2c indicates + 99 and + 3003 mutations 3 indicates + 126 + 130 and + 3187 mutations

2431


detected by 15 agarose gel electrophoresis stained withethidium bromide The presence of a fragment of 218 bpindicated the AluyHG absence and 540 bp the AluyHGpresence

Allele and genotype frequencies were estimated by directcount The adherence of the genotype frequencies under theassumption of the HardyndashWeinberg equilibrium was evalu-ated by using the Guo and Thompson exact test (Guo andThompson 1992) implemented in the Genepop 42 soft-ware (Rousset 2008) The LD pattern was evaluated by calcu-lating D0 LOD scores and LD plots using Haploview 42(Barrett et al 2005) considering only variation sites with aminimum allele frequency (MAF) of 1 Haplotypes wereinferred by probabilistic models using two distinct algorithmsPHASE 211 (Stephens et al 2001 Stephens and Donnelly2003) and Partition-Ligation Expectation Maximization (PL-EM) (Qin et al 2002) A Perl script named HaploRunner(available at wwwcastelli-labnet last accessed September16 2013) was used to perform independent runs and com-pare the results between both methods keeping only thehaplotypes that were equally inferred by PHASE and PL-EMThe script performed 10 runs for both PHASE and PL-EM andthe following configuration was used for PHASE randomseed values for each run the number of interactions was

set to 1000 thinning interval set to 1 and the burn-in valueset to 100 for PL-EM the parameters used were Top value setto zero Pair size value set to 2 Buffer set to 1300 and theRound value set to 200

The HLA-G coding and 30-UTR data from 14 different pop-ulations available in the 1000Genomes Project was directlydownloaded from the website (www1000genomesorg lastaccessed February 10 2013) (1000Genomes ProjectConsortium 2012) These data were converted into aGenepop format and a PED file (for Haploview) by usingPGDSpider version 2019 (Lischer and Excoffier 2012)Despite the HLA-G data from the 1000GenomesConsortium being already phased the same approach de-scribed above was used to infer haplotypes by concatenating1000Genome data and data from this article It was done inorder to have the same approach for all the samplesNevertheless the compatibility between the phased datafrom 1000Genomes Consortium and the method proposedabove was higher than 99

Acknowledgments

The authors thank Dr Paulo Cesar Ghedini for his invaluablehelp This work was supported by the Brazilian NationalResearch Council - CNPq (grant number 4708732011-6)

FIG 4 Network of the HLA-G 30-UTRAluyHG haplotypes considering data from Brazil Congo Senegal and France The network was calculated byMedian-Joining method (Bandelt et al 1999) using the Network Program version 4611 considering the Brazil Congo Senegal and France data andexcluding the haplotypes with frequencies lower than 1 The AluyHG1 allele indicates the absence of the AluyHG element whereas the AluyHG2allele indicates the presence of the AluyHG The HLA-G 30-UTR haplotypes were named according to earlier studies (Castelli et al 2010 Lucena-Silvaet al 2012) and different colors or shades of gray indicate different HLA-G lineages as indicated in figure 3

2432


and the binational collaborative research program CAPES-COFECUB (project number 65309)

References1000Genomes Project Consortium 2012 An integrated map of genetic

variation from 1092 human genomes Nature 49156ndash65Amiot L Ferrone S Grosse-Wilde H Seliger B 2011 Biology of HLA-G in

cancer a candidate molecule for therapeutic intervention Cell MolLife Sci 68417ndash431

Bandelt HJ Forster P Rohl A 1999 Median-joining networks for infer-ring intraspecific phylogenies Mol Biol Evol 1637ndash48

Barrett JC Fry B Maller J Daly MJ 2005 Haploview analysis andvisualization of LD and haplotype maps Bioinformatics 21263ndash265

Batzer MA Deininger PL 2002 Alu repeats and human genomic diver-sity Nat Rev Genet 3370ndash379

Berger DS Hogge WA Barmada MM Ferrell RE 2010 Comprehensiveanalysis of HLA-G implications for recurrent spontaneous abortionReprod Sci 17331ndash338

Carosella ED 2011 The tolerogenic molecule HLA-G Immunol Lett 13822ndash24

Carosella ED Moreau P Lemaoult J Rouas-Freiss N 2008 HLA-G frombiology to clinical benefits Trends Immunol 29125ndash132

Castelli EC Mendes-Junior CT Deghaide NH de Albuquerque RS MunizYC Simoes RT Carosella ED Moreau P Donadi EA 2010 Thegenetic structure of 30untranslated region of the HLA-G genepolymorphisms and haplotypes Genes Immun 11134ndash141

Castelli EC Mendes-Junior CT Donadi EA 2007 HLA-G alleles and HLA-G 14 bp polymorphisms in a Brazilian population Tissue Antigens 7062ndash68

Castelli EC Mendes-Junior CT Veiga-Castelli LC Roger M Moreau PDonadi EA 2011 A comprehensive study of polymorphic sitesalong the HLA-G gene implication for gene regulation and evolu-tion Mol Biol Evol 283069ndash3086

Castelli EC Moreau P Oya e Chiromatzo A Mendes-Junior CT Veiga-Castelli LC Yaghi L Giuliatti S Carosella ED Donadi EA 2009 Insilico analysis of microRNAS targeting the HLA-G 30 untranslatedregion alleles and haplotypes Hum Immunol 701020ndash1025

Castro MJ Morales P Martinez-Laso J Allende L Rojo-Amigo RGonzalez-Hevilla M Varela P Moreno A Garcia-Berciano MArnaiz-Villena A 2000 Evolution of MHC-G in humans and pri-mates based on three new 30UT polymorphisms Hum Immunol 611157ndash1163

Cervera I Herraiz MA Penaloza J Barbolla ML Jurado ML Macedo JVidart JA Martinez-Laso J 2010 Human leukocyte antigen-G allelepolymorphisms have evolved following three different evolutionarylineages based on intron sequences Hum Immunol 711109ndash1115

Cirulli V Zalatan J McMaster M Prinsen R Salomon DR Ricordi CTorbett BE Meda P Crisa L 2006 The class I HLA repertoire ofpancreatic islets comprises the nonclassical class Ib antigen HLA-GDiabetes 551214ndash1222

Colonna M 1997 Specificity and function of immunoglobulin super-family NK cell inhibitory and stimulatory receptors Immunol Rev155127ndash133

Contini P Ghio M Poggi A Filaci G Indiveri F Ferrone S Puppo F 2003Soluble HLA-A-B-C and -G molecules induce apoptosis in T andNKCD8( + ) cells and inhibit cytotoxic T cell activity through CD8ligation Eur J Immunol 33125ndash134

Courtin D Milet J Sabbagh A et al (13 co-authors) 2013 HLA-G 30

UTR-2 haplotype is associated with Human African trypanosomiasissusceptibility Infect Genet Evol 171ndash7

Crisa L McMaster MT Ishii JK Fisher SJ Salomon DR 1997Identification of a thymic epithelial cell subset sharing expressionof the class Ib HLA-G molecule with fetal trophoblasts J Exp Med186289ndash298

Crispim JC Duarte RA Soares CP Costa R Silva JS Mendes-Junior CTWastowski IJ Faggioni LP Saber LT Donadi EA 2008 Humanleukocyte antigen-G expression after kidney transplantation is

associated with a reduced incidence of rejection TransplImmunol 18361ndash367

Di Cristofaro J El Moujally D Agnel A Mazieres S Cortey M Basire AChiaroni J Picard C 2013 HLA-G haplotype structure shows goodconservation between different populations and good correlationwith high normal and low soluble HLA-G expression HumImmunol 74203ndash206

Donadi EA Castelli EC Arnaiz-Villena A Roger M Rey D Moreau P2011 Implications of the polymorphism of HLA-G on its functionregulation evolution and disease association Cell Mol Life Sci 68369ndash395

Dunn DS Choy MK Phipps ME Kulski JK 2007 The distribution ofmajor histocompatibility complex class I polymorphic Alu insertionsand their associations with HLA alleles in a Chinese population fromMalaysia Tissue Antigens 70136ndash143

Dunn DS Inoko H Kulski JK 2006 The association between non-melanoma skin cancer and a young dimorphic Alu elementwithin the major histocompatibility complex class I genomicregion Tissue Antigens 68127ndash134

Ferreira LB Mendes-Junior CT Wiezel CE Luizon MR Simoes AL 2006Genomic ancestry of a sample population from the state ofSao Paulo Brazil Am J Hum Biol 18702ndash705

Gao GF Willcox BE Wyer JR et al (11 co-authors) 2000 Classical andnonclassical class I major histocompatibility complex moleculesexhibit subtle conformational differences that affect binding toCD8alphaalpha J Biol Chem 27515232ndash15238

Garcia A Milet J Courtin D et al (11 co-authors) 2013 Association ofHLA-G 30UTR polymorphisms with response to malaria infection afirst insight Infect Genet Evol 16263ndash269

Gaudieri S Dawkins RL Habara K Kulski JK Gojobori T 2000 SNPprofile within the human major histocompatibility complex revealsan extreme and interrupted level of nucleotide diversity GenomeRes 101579ndash1586

Guo SW Thompson EA 1992 Performing the exact test ofHardyndashWeinberg proportion for multiple alleles Biometrics 48361ndash372

Henn BM Cavalli-Sforza LL Feldman MW 2012 The great humanexpansion Proc Natl Acad Sci U S A 10917758ndash17764

Hviid TV 2006 HLA-G in human reproduction aspects of geneticsfunction and pregnancy complications Hum Reprod Update 12209ndash232

Hviid TV Hylenius S Rorbye C Nielsen LG 2003 HLA-G allelic variantsare associated with differences in the HLA-G mRNA isoform profileand HLA-G mRNA levels Immunogenetics 5563ndash79

Hviid TV Rizzo R Christiansen OB Melchiorri L Lindhard A BaricordiOR 2004 HLA-G and IL-10 in serum in relation to HLA-G genotypeand polymorphisms Immunogenetics 56135ndash141

Hviid TV Rizzo R Melchiorri L Stignani M Baricordi OR 2006Polymorphism in the 50 upstream regulatory and 30 untranslatedregions of the HLA-G gene in relation to soluble HLA-G and IL-10expression Hum Immunol 6753ndash62

Hviid TV Sorensen S Morling N 1999 Polymorphism in the regulatoryregion located more than 11 kilobases 50 to the start site oftranscription the promoter region and exon 1 of the HLA-Ggene Hum Immunol 601237ndash1244

Ito T Ito N Saathoff M Stampachiacchiere B Bettermann A Bulfone-Paus S Takigawa M Nickoloff BJ Paus R 2005 Immunology of thehuman nail apparatus the nail matrix is a site of relative immuneprivilege J Invest Dermatol 1251139ndash1148

Jassem RM Shani WS Loisel DA Sharief M Billstrand C Ober C 2012HLA-G polymorphisms and soluble HLA-G protein levels in womenwith recurrent pregnancy loss from Basrah province in IraqHum Immunol 73811ndash817

Jurka J 1997 Sequence patterns indicate an enzymatic involvementin integration of mammalian retroposons Proc Natl Acad SciU S A 941872ndash1877

Jurka J Smith T 1988 A fundamental division in the Alufamily of repeated sequences Proc Natl Acad Sci U S A 854775ndash4778

2433


Klein J Sato A 2000 The HLA system First of two parts N Engl J Med343702ndash709

Kulski JK Dunn DS 2005 Polymorphic Alu insertions within the MajorHistocompatibility Complex class I genomic region a brief reviewCytogenet Genome Res 110193ndash202

Kulski JK Martinez P Longman-Jacobsen N Wang W Williamson JDawkins RL Shiina T Naruse T Inoko H 2001 The associationbetween HLA-A alleles and an Alu dimorphism near HLA-GJ Mol Evol 53114ndash123

Larsen MH Hviid TV 2009 Human leukocyte antigen-G polymorphismin relation to expression function and disease Hum Immunol 701026ndash1034

Le Discorde M Moreau P Sabatier P Legeais JM Carosella ED 2003Expression of HLA-G in human cornea an immune-privileged tissueHum Immunol 641039ndash1044

Lee N Malacko AR Ishitani A Chen MC Bajorath J Marquardt HGeraghty DE 1995 The membrane-bound and soluble forms ofHLA-G bind identical sets of endogenous peptides but differ withrespect to TAP association Immunity 3591ndash600

LeMaoult J Le Discorde M Rouas-Freiss N Moreau P Menier CMcCluskey J Carosella ED 2003 Biology and functions of humanleukocyte antigen-G in health and sickness Tissue Antigens 62273ndash284

Lischer HE Excoffier L 2012 PGDSpider an automated data conversiontool for connecting population genetics and genomics programsBioinformatics 28298ndash299

Lucena-Silva N Monteiro AR de Albuquerque RS Gomes RG Mendes-Junior CT Castelli EC Donadi EA 2012 Haplotype frequencies basedon eight polymorphic sites at the 30 untranslated region of the HLA-G gene in individuals from two different geographical regions ofBrazil Tissue Antigens 79272ndash278

Mallet V Blaschitz A Crisa L Schmitt C Fournel S King A Loke YWDohr G Le Bouteiller P 1999 HLA-G in the human thymus asubpopulation of medullary epithelial but not CD83( + ) dendriticcells expresses HLA-G as a membrane-bound and soluble proteinInt Immunol 11889ndash898

Manaster I Goldman-Wohl D Greenfield C Nachmani D Tsukerman PHamani Y Yagel S Mandelboim O 2012 MiRNA-mediated controlof HLA-G expression and function PLoS One 7e33395

Martelli-Palomino G Pancoto JA Muniz YCN et al (13 co-authors)Forthcoming 2013 Polymorphic sites at the 30 untranslated regionof the HLA-G gene are associated with differential HLA-G solublelevels in the Brazilian and French population PLoS One

Martinez-Laso J Herraiz MA Penaloza J Barbolla ML Jurado MLMacedo J Vidart J Cervera I 2013 Promoter sequences confirmthe three different evolutionary lineages described for HLA-G HumImmunol 74383ndash388

Menier C Rabreau M Challier JC Le Discorde M Carosella ED Rouas-Freiss N 2004 Erythroblasts secrete the nonclassical HLA-Gmolecule from primitive to definitive hematopoiesis Blood 1043153ndash3160

Moreau P Flajollet S Carosella ED 2009 Non-classical transcriptionalregulation of HLA-G an update J Cell Mol Med 132973ndash2989

Muniz YC Ferreira LB Mendes-Junior CT Wiezel CE Simoes AL 2008Genomic ancestry in urban Afro-Brazilians Ann Hum Biol 35104ndash111

Ober C Aldrich CL 1997 HLA-G polymorphisms neutral evolution ornovel function J Reprod Immunol 361ndash21

Ponte M Cantoni C Biassoni R Tradori-Cappai A Bentivoglio G VitaleC Bertone S Moretta A Moretta L Mingari MC 1999 Inhibitoryreceptors sensing HLA-G1 molecules in pregnancy decidua-associated natural killer cells express LIR-1 and CD94NKG2A andacquire p49 an HLA-G1-specific receptor Proc Natl Acad Sci U S A965674ndash5679

Qin ZS Niu T Liu JS 2002 Partition-ligation-expectation-maximizationalgorithm for haplotype inference with single-nucleotide polymor-phisms Am J Hum Genet 711242ndash1247

Rajagopalan S Long EO 1999 A human histocompatibility leukocyteantigen (HLA)-G-specific receptor expressed on all natural killercells J Exp Med 1891093ndash1100

Rizzo R Bortolotti D Fredj NB et al (14 co-authors) 2012 Role of HLA-G 14bp deletioninsertion and + 3142CgtG polymorphisms in theproduction of sHLA-G molecules in relapsing-remitting multiplesclerosis Hum Immunol 731140ndash1146

Rousseau P Le Discorde M Mouillot G Marcou C Carosella ED MoreauP 2003 The 14 bp deletion-insertion polymorphism in the 30 UTregion of the HLA-G gene influences HLA-G mRNA stability HumImmunol 641005ndash1010

Rousset F 2008 genepoprsquo007 a complete re-implementation of thegenepop software for Windows and Linux Mol Ecol Resour 8103ndash106

Rowold DJ Herrera RJ 2000 Alu elements and the human genomeGenetica 10857ndash72

Sabbagh A Courtin D Milet J et al (11 co-authors) 2013 Association ofHLA-G 30 untranslated region polymorphisms with antibody re-sponse against Plasmodium falciparum antigens preliminary resultsTissue Antigens 8253ndash58

Shiroishi M Kuroki K Rasubala L Tsumoto K Kumagai I Kurimoto EKato K Kohda D Maenaka K 2006 Structural basis for recognitionof the nonclassical MHC molecule HLA-G by the leukocyte Ig-likereceptor B2 (LILRB2LIR2ILT4CD85d) Proc Natl Acad Sci U S A10316412ndash16417

Silva TG Crispim JC Miranda FA Hassumi MK de Mello JM Simoes RTSouto F Soares EG Donadi EA Soares CP 2011 Expression ofthe nonclassical HLA-G and HLA-E molecules in laryngeal lesionsas biomarkers of tumor invasiveness Histol Histopathol 261487ndash1497

Solberg OD Mack SJ Lancaster AK Single RM Tsai Y Sanchez-Mazas AThomson G 2008 Balancing selection and heterogeneity acrossthe classical human leukocyte antigen loci a meta-analytic reviewof 497 population studies Hum Immunol 69443ndash464

Solier C Mallet V Lenfant F Bertrand A Huchenq A Le Bouteiller P2001 HLA-G unique promoter region functional implicationsImmunogenetics 53617ndash625

Stephens M Donnelly P 2003 A comparison of bayesian methods forhaplotype reconstruction from population genotype data AmJ Hum Genet 731162ndash1169

Stephens M Smith NJ Donnelly P 2001 A new statistical method forhaplotype reconstruction from population data Am J Hum Genet68978ndash989

Svendsen SG Hantash BM Zhao L Faber C Bzorek M Nissen MH HviidTV 2013 The expression and functional activity of membrane-bound human leukocyte antigen-G1 are influenced by the30-untranslated region Hum Immunol 74818ndash827

Tan Z Randall G Fan J et al (11 co-authors) 2007 Allele-specific tar-geting of microRNAs to HLA-G and risk of asthma Am J Hum Genet81829ndash834

Tan Z Shon AM Ober C 2005 Evidence of balancing selection at theHLA-G promoter region Hum Mol Genet 143619ndash3628

Tian W Wang F Cai JH Li LX 2008 Polymorphic insertions in 5 Alu lociwithin the major histocompatibility complex class I region and theirlinkage disequilibria with HLA alleles in four distinct populations inmainland China Tissue Antigens 72559ndash567

Yie SM Li LH Xiao R Librach CL 2008 A single base-pair mutationin the 30-untranslated region of HLA-G mRNA is associated withpre-eclampsia Mol Hum Reprod 14649ndash653

Zietkiewicz E Richer C Makalowski W Jurka J Labuda D 1994 A youngAlu subfamily amplified independently in human and African greatapes lineages Nucleic Acids Res 225608ndash5612

2434


Le Discorde et al 2003 Menier et al 2004 Ito et al 2005 Cirulliet al 2006) 2) the presence of several membrane and solubleisoforms due to alternative splicing of the full-length HLA-GmRNA (reviewed at [Hviid 2006 Donadi et al 2011]) 3) aunique short cytoplasmic tail due to the presence of a pre-mature stop codon at exon 6 (Hviid 2006 Donadi et al 2011)4) limited protein variability (Hviid 2006 Larsen and Hviid2009 Donadi et al 2011) 5) a unique 50URR (50 upstreamregulatory region) (Solier et al 2001 Moreau et al 2009) and6) a very polymorphic 50 promoter (Hviid et al 1999 2004Tan et al 2005 Hviid et al 2006 Castelli et al 2011 Martinez-Laso et al 2013) and 30-untranslated region (30-UTR) (Castelliet al 2010)

In pathological conditions HLA-G expression has beendetected in tumors chronic viral infections inflammatoryand autoimmune diseases and in engrafted tissues(LeMaoult et al 2003 Carosella et al 2008 Crispim et al2008 Amiot et al 2011 Silva et al 2011) In this contextthe expression of HLA-G in chronic inflammation autoim-mune diseases and allotransplants has been associated with abetter prognosis due to the inhibition of the immuneresponse In contrast the inhibition of the immune responsemay be harmful in chronic viral infections and tumorssituations in which a vigorous immune response would berequired (Carosella 2011)

In contrast to the classical HLA class I genes limited var-iability at the HLA-G coding region has been observed inworldwide populations (reviewed by Donadi et al [2011]and Larsen and Hviid [2009]) According to theInternational ImmunoGeneTics Database (IMGTDatabasedatabase version 3120 April 2013) 50 HLA-G alleles (or dif-ferent coding haplotypes) are currently described generating16 different full-length proteins Of these five alleles are fre-quently observed in worldwide populations and they wouldbe considered as polymorphisms (Castelli et al 2007 2011Donadi et al 2011) The low variability at the coding region isprobably associated with the very limited repertoire of pep-tides that HLA-G would present to T cells and also to ensurea nonvariable molecule structure permitting the modulationof the immune response in vital situations such as pregnancyIndeed it has been considered that purifying selection hasshaped the HLA-G coding region variability avoiding a highdegree of variation (Castro et al 2000) In contrast a relativelyhigher degree of variation has been observed at the HLA-G50URR (Hviid et al 1999 Tan et al 2005 Hviid et al 2006Castelli et al 2011 Martinez-Laso et al 2013) and 30-UTR(Castelli et al 2010) which may be highly implicated inHLA-G expression regulation (Hviid et al 2006 Donadi et al2011) mRNA stability (Hviid et al 2003 Rousseau et al 2003)mRNA degradation (Tan et al 2007 Yie et al 2008) andpossible differential activity of microRNAs (miRNA) (Tanet al 2007 Castelli et al 2009 Manaster et al 2012)

The HLA-G variability in the regulatory coding and 30UTregion has already been evaluated in a Brazilian admixedpopulation (Castelli et al 2011) This study together withothers that evaluated the promoter region indicated thatthe HLA-G gene presents a high linkage disequilibrium (LD)along the entire gene exhibiting few extended haplotypes and

a haplotype pattern that seems to be the same worldwide(Ober and Aldrich 1997 Hviid et al 1999 2004 Tan et al 2005Hviid et al 2006 Larsen and Hviid 2009 Cervera et al 2010Castelli et al 2011 Donadi et al 2011 Lucena-Silva et al 2012Courtin et al 2013 Di Cristofaro et al 2013 Garcia et al 2013Martinez-Laso et al 2013 Sabbagh et al 2013)

In humans Alu sequences are the most abundantelements of the SINE (Short Interspersed Nuclear Element)retrotransposon family presenting approximately 300 bp(Rowold and Herrera 2000) Alu retroelements have beenamplified within the human genome during recent evolution-ary period and are useful polymorphic markers for humanpopulation origin studies (Batzer and Deininger 2002 Kulskiand Dunn 2005) These elements have been classified intodifferent subfamilies (AluJ AluS and AluY) based on theirmutation pattern genetic age and sequence difference(Jurka and Smith 1988 Batzer and Deininger 2002) Amongthe AluY subfamilies the AluYb8 is a human-specific poly-morphic element that has been used as a relevant toolfor population studies Five different AluYb8 sequenceshave already been identified in the human MHC class Iregion ie AluyMICB AluyTF AluyHJ AluyHF and AluyHG(Kulski and Dunn 2005)

Among the features that turn the AluyHG element into aninteresting marker for population and HLA-G studies are 1) itbelongs to the AluYb8 subfamily and is located approximately20 kb downstream from the 30-UTR of the HLA-G gene (Kulskiet al 2001) 2) this mobile element is dimorphic ie is eitherpresent or absent (Rowold and Herrera 2000) 3) it exhibitsidentity for descendants ie if two individuals share an inser-tion at the same locus they have a common ancestor (Rowoldand Herrera 2000) 4) this marker is not fixed in the humangenome ie it is still polymorphic (Kulski and Dunn 2005)5) its ancestral state is known ie the element absence isthe original state (Rowold and Herrera 2000) and 6) theAluyHG element evaluation is straightforward and simpleFurthermore the AluYb8 subfamily seems to be transposi-tionally active and seems to be a human-specific fragment(Zietkiewicz et al 1994) The AluYb8 retrotranspositionmechanism involves the transposition of a RNA polymeraseIII retrotranscribed product probably by a reverse transcrip-tase encoded into a LINE sequence (Long InterspersedNuclear Element) because the Alu elements do not presentencoded reverse transcriptase (Batzer and Deininger 2002)

To better understand the evolutionary mechanisms actingon the HLA-G gene the presence or absence of the AluyHGelement was evaluated associating this Alu site with othervariation sites at the coding 30-UTR or both regions in fourdistinct populations (Brazilian French Congolese andSenegalese) To evaluate whether the pattern observed byus was also present worldwide we compared our resultswith those available at the 1000Genomes Consortium data(1000Genomes Project Consortium 2012)

ResultsThe presence or absence of the AluyHG element was evalu-ated in 641 individuals 165 Brazilians (BRA) 161 Congolese(CNG) 193 Senegalese (SEE) and 122 French (FRE) The

2424








Africa


Asia




America






AluyHGallele

Frequencies


N 152b 161 193 122

UTR-1 Present 02500 01056 01036 02131

UTR-1 Absent 00230 00342 00052 00533

UTR-2 Absent 02500 02112 03575 02664


UTR-3 Absent 01250 03043 02798 01352

UTR-4 Absent 01282 01056 00518 01434

UTR-5 Absent 00757 00932 01321 00369

UTR-6 Absent 01020 01304 00648 00861








2425







2426


Tab

le3

Freq

uen

cyof

the

HLA

-GC

odin

g30

-UT

RH

aplo

typ

esin

Wor

ldw

ide

Popu

lati

on

Euro

pe

Asi

aA

fric

aA

mer

ica

Pop

ula

tion


Finnish(Finland)

Iberian(Spain)

Tuscany(Italy)



Japanese

(TokyoJapan)


Luhya

(WebuyeKenya)


Colombian(Medellin

Colombia)

MexicanAncestry

(LosAngelisUSA)

EuropeanAncestry

(UtahUSA)

AfricanAncestry

(SouthwestUSA)

Braziliansa


HLA

-G30

-UT

RH

aplo

typ

esb

HLA

-GC

odin

gH

aplo

typ

esc

N89

9314

9810

097

8988

9755

6066

8561

108

UT

R-1

G0

101

01

010

3371

036

020

3214

029

080

4250

029

470

2619

012

180

2391

027

780

2373

027

270

3941

021

820

2360

G0

101

01

04mdash

mdashmdash

mdashmdash

mdashmdash

002

560

0109

000

93mdash

000

76mdash

000

91mdash

G0

101

03

G0

101

01

01d

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mdashmdash

000

50mdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

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UT

R-2

G0

101

02

010

2303

017

740

3571

020

410

0400

011

050

1607

014

100

1848

011

110

1780

021

270

2059

020

910

1530

G0

101

02

02mdash

mdashmdash

000

51mdash

mdashmdash

000

640

0380

mdashmdash

mdashmdash

mdashmdash

G0

106

006

180

0269

003

570

0663

001

000

0263

000

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000

540

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004

240

0227

004

710

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005

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01

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0108

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001

500

0421

000

600

0962

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43mdash

000

850

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000

590

0636

004

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01

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(+18

8C

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001

53mdash

000

53mdash

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280

0217

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52mdash

001

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101

03

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01

010

201

d0

0056

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82mdash

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000

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R-3

G0

104

01

004

490

0591

007

140

0969

025

500

2474

045

240

0256

001

090

1296

017

800

1515

006

470

0364

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60G

01

040

3mdash

mdashmdash

mdash0

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000

530

0179

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104

04

001

120

0054

mdash0

0306

mdashmdash

mdash0

2244

009

780

0556

003

390

0076

002

350

1000

003

20G

01

040

5mdash

mdashmdash

mdashmdash

mdashmdash

000

640

0109

000

930

0169

mdashmdash

000

91mdash

G0

101

08

mdashmdash

mdashmdash

mdash0

0053

001

19mdash

mdashmdash

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01

040

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188

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mdashmdash

mdashmdash

mdashmdash

mdash0

0064

mdashmdash

mdashmdash

mdashmdash

mdashG

01

010

3G

01

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1dmdash

mdashmdash

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001

580

0119

mdashmdash

mdashmdash

mdashmdash

mdashmdash

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R-4

G0

101

01

050

1180

028

490

1071

014

290

0200

004

740

0060

011

540

0761

012

960

1356

007

580

1529

005

450

0970

G0

101

09

mdashmdash

mdashmdash

mdashmdash

mdash0

0321

002

72mdash

000

850

0076

mdash0

0091

000

50

UT

R-5

G0

103

002

810

0161

mdash0

0306

mdash0

0263

001

790

0897

008

700

1111

007

630

0985

003

530

1364

008

80G

01

040

10

0056

mdashmdash

000

51mdash

mdashmdash

mdashmdash

000

93mdash

mdashmdash

mdashmdash

G0

101

08

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mdashmdash

mdashmdash

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000

90

(con

tin

ued)

2427


Tab

le3

Con

tin

ued

Euro

pe

Asi

aA

fric

aA

mer

ica

Pop

ula

tion


Finnish(Finland)

Iberian(Spain)

Tuscany(Italy)



Japanese

(TokyoJapan)


Luhya

(WebuyeKenya)


Colombian(Medellin

Colombia)

MexicanAncestry

(LosAngelisUSA)

EuropeanAncestry

(UtahUSA)

AfricanAncestry

(SouthwestUSA)

Braziliansa


HLA

-G30

-UT

RH

aplo

typ

esb

HLA

-GC

odin

gH

aplo

typ

esc

N89

9314

9810

097

8988

9755

6066

8561

108

UT

R-6

G0

101

01

040

0618

001

080

0714

002

04mdash

mdashmdash

008

330

1359

008

330

0678

003

790

0118

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0059

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101

01

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101

01

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1019

C)d

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0051

mdashmdash

mdashmdash

mdashmdash

mdashmdash

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UT

R-7

G0

101

03

010

0899

004

840

0357

004

590

2200

016

840

0471

mdashmdash

004

630

0085

004

550

0471

000

910

0370

G0

101

05

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

000

50

UT

R-8

G0

106

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

001

40

UT

R-9

G0

101

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mdashmdash

mdashmdash

mdashmdash

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8fG

01

010

201

mdashmdash

mdashmdash

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mdashmdash

mdashmdash

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0076

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9fG

01

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301

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000

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NO

TEmdash

N=

num

ber

ofin

divi

dual

sa Br

azili

ans

from

Rib

eira

oPr

eto

Stat

eof

Sao

Paul

oBr

azil

The

HLA

-Gva

riabi

lity

was

prev

ious

pub

lishe

dby

Cas

telli

etal

(2

011)

bH

LA-G

30-U

TR

hapl

otyp

esw

ere

nam

edac

cord

ing

toC

aste

lliet

al

(201

0)an

dLu

cena

-Silv

aet

al

(201

2)

c HLA

-Gco

din

gha

plo

typ

esw

ere

nam

edac

cord

ing

toth

eIn

tern

atio

nal

Imm

unog

enet

ics

Dat

abas

e(I

MG

TH

LA)

dR

ecom

bina

ntha

plot

ypes

e Li

kely

ance

stor

alle

lefo

llow

edby

the

mut

atio

nth

atde

fined

this

hapl

otyp

ef T

his

30-U

TR

hap

loty

pes

was

not

dete

cted

inth

est

udie

sby

Cas

telli

etal

(2

010)

and

Luce

na-

Silv

aet

al

(201

2)

2428










2429











2430






2431






Acknowledgments



2432













































2433







































2434








Africa


Asia




America






AluyHGallele

Frequencies


N 152b 161 193 122

UTR-1 Present 02500 01056 01036 02131

UTR-1 Absent 00230 00342 00052 00533

UTR-2 Absent 02500 02112 03575 02664


UTR-3 Absent 01250 03043 02798 01352

UTR-4 Absent 01282 01056 00518 01434

UTR-5 Absent 00757 00932 01321 00369

UTR-6 Absent 01020 01304 00648 00861








2425







2426


Tab

le3

Freq

uen

cyof

the

HLA

-GC

odin

g30

-UT

RH

aplo

typ

esin

Wor

ldw

ide

Popu

lati

on

Euro

pe

Asi

aA

fric

aA

mer

ica

Pop

ula

tion


Finnish(Finland)

Iberian(Spain)

Tuscany(Italy)



Japanese

(TokyoJapan)


Luhya

(WebuyeKenya)


Colombian(Medellin

Colombia)

MexicanAncestry

(LosAngelisUSA)

EuropeanAncestry

(UtahUSA)

AfricanAncestry

(SouthwestUSA)

Braziliansa


HLA

-G30

-UT

RH

aplo

typ

esb

HLA

-GC

odin

gH

aplo

typ

esc

N89

9314

9810

097

8988

9755

6066

8561

108

UT

R-1

G0

101

01

010

3371

036

020

3214

029

080

4250

029

470

2619

012

180

2391

027

780

2373

027

270

3941

021

820

2360

G0

101

01

04mdash

mdashmdash

mdashmdash

mdashmdash

002

560

0109

000

93mdash

000

76mdash

000

91mdash

G0

101

03

G0

101

01

01d

mdashmdash

mdashmdash

000

50mdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdash

UT

R-2

G0

101

02

010

2303

017

740

3571

020

410

0400

011

050

1607

014

100

1848

011

110

1780

021

270

2059

020

910

1530

G0

101

02

02mdash

mdashmdash

000

51mdash

mdashmdash

000

640

0380

mdashmdash

mdashmdash

mdashmdash

G0

106

006

180

0269

003

570

0663

001

000

0263

000

60mdash

000

540

0278

004

240

0227

004

710

0091

005

10G

01

05N

mdash0

0108

mdash0

0255

001

500

0421

000

600

0962

005

43mdash

000

850

0303

000

590

0636

004

20G

01

05N

(+18

8C

)emdash

mdashmdash

001

53mdash

000

53mdash

001

280

0217

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0085

001

52mdash

001

82mdash

G0

101

03

01G

01

010

201

d0

0056

mdashmdash

mdashmdash

mdash0

0060

mdashmdash

mdashmdash

mdashmdash

001

82mdash

G0

101

14

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

000

50

UT

R-3

G0

104

01

004

490

0591

007

140

0969

025

500

2474

045

240

0256

001

090

1296

017

800

1515

006

470

0364

004

60G

01

040

3mdash

mdashmdash

mdash0

0050

000

530

0179

mdashmdash

mdashmdash

mdashmdash

mdash0

0050

G0

104

04

001

120

0054

mdash0

0306

mdashmdash

mdash0

2244

009

780

0556

003

390

0076

002

350

1000

003

20G

01

040

5mdash

mdashmdash

mdashmdash

mdashmdash

000

640

0109

000

930

0169

mdashmdash

000

91mdash

G0

101

08

mdashmdash

mdashmdash

mdash0

0053

001

19mdash

mdashmdash

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mdashmdash

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01

040

4(+

188

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mdashmdash

mdashmdash

mdashmdash

mdash0

0064

mdashmdash

mdashmdash

mdashmdash

mdashG

01

010

3G

01

040

1dmdash

mdashmdash

mdash0

0050

001

580

0119

mdashmdash

mdashmdash

mdashmdash

mdashmdash

UT

R-4

G0

101

01

050

1180

028

490

1071

014

290

0200

004

740

0060

011

540

0761

012

960

1356

007

580

1529

005

450

0970

G0

101

09

mdashmdash

mdashmdash

mdashmdash

mdash0

0321

002

72mdash

000

850

0076

mdash0

0091

000

50

UT

R-5

G0

103

002

810

0161

mdash0

0306

mdash0

0263

001

790

0897

008

700

1111

007

630

0985

003

530

1364

008

80G

01

040

10

0056

mdashmdash

000

51mdash

mdashmdash

mdashmdash

000

93mdash

mdashmdash

mdashmdash

G0

101

08

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

000

90

(con

tin

ued)

2427


Tab

le3

Con

tin

ued

Euro

pe

Asi

aA

fric

aA

mer

ica

Pop

ula

tion


Finnish(Finland)

Iberian(Spain)

Tuscany(Italy)



Japanese

(TokyoJapan)


Luhya

(WebuyeKenya)


Colombian(Medellin

Colombia)

MexicanAncestry

(LosAngelisUSA)

EuropeanAncestry

(UtahUSA)

AfricanAncestry

(SouthwestUSA)

Braziliansa


HLA

-G30

-UT

RH

aplo

typ

esb

HLA

-GC

odin

gH

aplo

typ

esc

N89

9314

9810

097

8988

9755

6066

8561

108

UT

R-6

G0

101

01

040

0618

001

080

0714

002

04mdash

mdashmdash

008

330

1359

008

330

0678

003

790

0118

010

000

0740

G0

101

01

01mdash

mdashmdash

000

51mdash

mdashmdash

mdashmdash

mdashmdash

mdash0

0059

mdash0

0090

G0

101

01

05mdash

mdashmdash

000

51mdash

mdashmdash

001

28mdash

mdashmdash

mdashmdash

mdash0

0149

G0

101

01

04(+

1019

C)d

mdashmdash

mdash0

0051

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdash

UT

R-7

G0

101

03

010

0899

004

840

0357

004

590

2200

016

840

0471

mdashmdash

004

630

0085

004

550

0471

000

910

0370

G0

101

05

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

000

50

UT

R-8

G0

106

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

001

40

UT

R-9

G0

101

08

000

56mdash

mdash0

0051

mdash0

0053

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdash

UT

R-1

8fG

01

010

201

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdash0

0076

mdashmdash

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010)

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(201

2)

2428










2429











2430






2431






Acknowledgments



2432













































2433







































2434







2426


Tab

le3

Freq

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MexicanAncestry

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EuropeanAncestry

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AfricanAncestry

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tin

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2427


Tab

le3

Con

tin

ued

Euro

pe

Asi

aA

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aA

mer

ica

Pop

ula

tion


Finnish(Finland)

Iberian(Spain)

Tuscany(Italy)



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(WebuyeKenya)


Colombian(Medellin

Colombia)

MexicanAncestry

(LosAngelisUSA)

EuropeanAncestry

(UtahUSA)

AfricanAncestry

(SouthwestUSA)

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azil

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lity

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ious

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telli

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LA-G

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his

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TR

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loty

pes

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est

udie

sby

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telli

etal

(2

010)

and

Luce

na-

Silv

aet

al

(201

2)

2428










2429











2430






2431






Acknowledgments



2432













































2433







































2434


Tab

le3

Freq

uen

cyof

the

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pe

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aA

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ica

Pop

ula

tion


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Iberian(Spain)

Tuscany(Italy)



Japanese

(TokyoJapan)


Luhya

(WebuyeKenya)


Colombian(Medellin

Colombia)

MexicanAncestry

(LosAngelisUSA)

EuropeanAncestry

(UtahUSA)

AfricanAncestry

(SouthwestUSA)

Braziliansa


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tin

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2427


Tab

le3

Con

tin

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Euro

pe

Asi

aA

fric

aA

mer

ica

Pop

ula

tion


Finnish(Finland)

Iberian(Spain)

Tuscany(Italy)



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(TokyoJapan)


Luhya

(WebuyeKenya)


Colombian(Medellin

Colombia)

MexicanAncestry

(LosAngelisUSA)

EuropeanAncestry

(UtahUSA)

AfricanAncestry

(SouthwestUSA)

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typ

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dual

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azili

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eira

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eof

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oBr

azil

The

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lity

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ious

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lishe

dby

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telli

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(2

011)

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LA-G

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TR

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his

30-U

TR

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loty

pes

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not

dete

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est

udie

sby

Cas

telli

etal

(2

010)

and

Luce

na-

Silv

aet

al

(201

2)

2428










2429











2430






2431






Acknowledgments



2432













































2433







































2434


Tab

le3

Con

tin

ued

Euro

pe

Asi

aA

fric

aA

mer

ica

Pop

ula

tion


Finnish(Finland)

Iberian(Spain)

Tuscany(Italy)



Japanese

(TokyoJapan)


Luhya

(WebuyeKenya)


Colombian(Medellin

Colombia)

MexicanAncestry

(LosAngelisUSA)

EuropeanAncestry

(UtahUSA)

AfricanAncestry

(SouthwestUSA)

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RH

aplo

typ

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097

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9755

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R-8

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40

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R-9

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R-1

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NO

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num

ber

ofin

divi

dual

sa Br

azili

ans

from

Rib

eira

oPr

eto

Stat

eof

Sao

Paul

oBr

azil

The

HLA

-Gva

riabi

lity

was

prev

ious

pub

lishe

dby

Cas

telli

etal

(2

011)

bH

LA-G

30-U

TR

hapl

otyp

esw

ere

nam

edac

cord

ing

toC

aste

lliet

al

(201

0)an

dLu

cena

-Silv

aet

al

(201

2)

c HLA

-Gco

din

gha

plo

typ

esw

ere

nam

edac

cord

ing

toth

eIn

tern

atio

nal

Imm

unog

enet

ics

Dat

abas

e(I

MG

TH

LA)

dR

ecom

bina

ntha

plot

ypes

e Li

kely

ance

stor

alle

lefo

llow

edby

the

mut

atio

nth

atde

fined

this

hapl

otyp

ef T

his

30-U

TR

hap

loty

pes

was

not

dete

cted

inth

est

udie

sby

Cas

telli

etal

(2

010)

and

Luce

na-

Silv

aet

al

(201

2)

2428










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Acknowledgments



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Acknowledgments



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Acknowledgments



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Acknowledgments



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Acknowledgments



2432













































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insights on the hla-g evolutionary history provided by a nearby alu insertion

Documents