combinatorial content of ccl3l ccl4l copy numbers ...copy numbers inﬂuence hiv-aids susceptibility...

C

Combinatorial content o
f CCL3L and CCL4L genecopy numbers influence HIV-AIDS susceptibility
in Ukrainian children

Ludmila Shostakovich-Koretskayaa,M, Gabriel Catanob,c,M,

Zoya A. Chykarenkoa,M, Weijing Heb,c, German Gornalusseb,c,

Srinivas Mummidib,c, Racquel Sanchezb,c, Matthew J. Doland,e,

Seema S. Ahujab,c, Robert A. Clarkb,c, Hemant Kulkarnib,c

and Sunil K. Ahujab,c,f

opyright © L

aDepartment of GUkraine, bVeterancDepartment of MCenter (SAMMC),States Air Force MUniversity of Texa

Correspondence toHIV/AIDS Center,

Tel: +1 210 567 0�

L.S.-K., G.C. and

Received: 25 Sept

DOI:10.1097/QAD

ISS

Objective: CCL3L and CCL4L genes encode HIV-suppressive chemokines, colocalize onchromosome 17q12 and have copy number variation. Copy number variation of CCL3Lassociates with HIV-AIDS susceptibility. Here, we determined the influence of thecombinatorial content of distinct CCL3L and CCL4L genes on HIV-AIDS susceptibility.

Methods: By designing gene-specific assays, the association between doses of all CCL3LorCCL4Lgenesor their individual duplicated components (CCL3La/b andCCL4La/b) withHIV-AIDS susceptibility was determined in 298 perinatally exposed Ukrainian children.

Results: The odds of transmission was increased in children with less than two copies ofCCL3L or CCL4L, compared with those with at least two copies, and 10-fold higherwhen both mother and offspring had less than two CCL3L or CCL4L copies, comparedwith mother–child pairs with at least two copies. The extent of the pair-wise corre-lations between CCL3La, CCL3Lb, CCL4La and CCL4Lb copy number varied exten-sively, with an inverse correlation between CCL4L genes that transcribe a classicalchemokine (CCL4La) versus aberrantly-spliced transcripts (CCL4Lb). Children posses-sing only CCL4Lb progressed four times faster to AIDS than those with only CCL4La. Alower content of CCL3L and CCL4L genes that transcribe classical chemokines wasassociated with enhanced HIV-AIDS susceptibility.

Conclusion: Transmission risk is greatest when mother and offspring both have lowCCL3L or CCL4L gene doses. The impact on HIV-AIDS susceptibility of the chemokinegene-rich locus on 17q12 is dependent on the balance between the doses of genesconferring protective (CCL3La and CCL4La) versus detrimental (CCL4Lb) effects. Hence,the combinatorial genomic content of distinct genes within a copy number variableregion may determine disease susceptibility.

� 2009 Wolters Kluwer Health | Lippincott Williams & Wilkins

AIDS 2009, 23:679–688

Keywords: AIDS, CCL3L, CCL4L, HIV, transmission

ippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.

eneral Pediatrics and Pediatric Infectious Diseases, Dnepropetrovsk State Medical Academy, Dnepropetrovsk,s Administration Research Center for AIDS and HIV-1 Infection, South Texas Veterans Healthcare System,edicine, University of Texas Health Science Center, dInfectious Disease Service, San Antonio Military MedicalBrooke Army Medical Center (BAMC), Fort Sam Houston, eHenry M. Jackson Foundation, Wilford Hall Unitededical Center, Lackland Air Force Base, and fDepartments of Microbiology and Immunology, and Biochemistry,s Health Science Center, San Antonio, Texas, USA.

Sunil K. Ahuja, Professor of Medicine and Microbiology and Immunology, Director, Veterans AdministrationUniversity of Texas Health Science Center, 7703 Floyd Curl Drive, San Antonio, TX 78229, USA.

233; fax: +1 210 567 0449; e-mail: [email protected]

Z.A.C. contributed equally to this article.

ember 2008; revised: 11 November 2008; accepted: 1 December 2008.

.0b013e3283270b3f

N 0269-9370 Q 2009 Wolters Kluwer Health | Lippincott Williams & Wilkins 679

mailto:[email protected]

http://dx.doi.org/10.1097/QAD.0b013e3283270b3f

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680

Introduction

AIDS 2009, Vol 23 No 6

The results of the recent STEP trial [1] showing that a HIV-1 vaccine may have increased the risk of acquiring HIVprompted a call to define with greater precision thecorrelates of protection. A powerful approach to accom-plish this is to identify host genetic determinants that alterrisk of HIV acquisition and disease progression rates.Identification of such determinants in children exposedperinatally to HIV can be especially informative becauseapproximately 70–90% of such children resist infection,despite high exposure to virus and absence of antiretroviralprophylaxis [2]. Here, we investigated such children todetermine the impact on HIV-AIDS susceptibility ofduplicated genomic regions that are enriched forchemokine genes for whom in-vitro studies [3–10]implicate a central role for their encoded products inHIV-AIDS pathogenesis.

Copy numbers of DNA segments [denoted as copynumber variations (CNVs)] are implicated in diseasesusceptibility [11–16]. A CNV with relevance to HIV-AIDS susceptibility is for the segmental duplication thatcontains the gene encoding CC chemokine ligand 3-like 1(CCL3L1). CCL3L1 [macrophage inflammatory protein1 alpha P (MIP-1aP)], a nonallelic isoform of CCL3(MIP-1aS), is the most potent agonist of CC chemokinereceptor 5 (CCR5), the major HIV coreceptor, and amongCCR5 chemokine ligands it exhibits maximal HIV-suppressive properties [5–8]. Association studies forthe D32 and other polymorphisms in CCR5 [17–23]established a key role for CCR5 expression in HIVpathogenesis. By analogy, the genotype–phenotypeassociations for the copy number of the CCL3L1-containing segmental duplication suggest a role for thisstructural variation in HIV-AIDS pathogenesis. Priorstudies indicate that a low copy number of CCL3L1 isassociatedwith an increased riskof acquiringHIV infection[24–28], higher viral loads [24,26,29,30], a faster rate ofdecline of CD4þ T-cell counts or progression to AIDS[24,30,31], impaired recovery of CD4þ T-cell counts [31]and function [30] during antiretroviral therapy (ART),lower HIV-specific CD4þ and CD8þ T-cell responses[29], reduced CCL3 chemokine levels [24,25,32], highernumbers of HIV target cells (%CD4þ/CCR5þ cells)[24,33] and reduced cell-mediated immune responses [30].Extending this concept to nonhuman primates, a lowcopy number of CCL3L genes in Asian and Chinesemacaques is associated with an accelerated progression toexperimental AIDS in these animals [34].

However, these previous studies have not accounted forthe full impact on HIV-AIDS susceptibility of CNV ofother chemokine genes that along with CCL3L1 localizeto chromosome 17q12, a hot spot for segmentalduplications [11,12]. These genes include two otherCCL3L (CCL3L2 and CCL3L3) genes and two CCL4L(MIP-1b-like) genes designated as CCL4L1 and

pyright © Lippincott Williams & Wilkins. Unauthor

CCL4L2 [3,4,9,10,32]. One possibility was that if theCNV of all the CCL3L and CCL4L genes reside on asingle segmental duplication, then the associations ofthese CNVs with HIV-AIDS susceptibility should beidentical. However, this is unlikely because this genomicregion is replete with many potential breakpoints thatmay lead to nonidentical duplicated segments [36].Underscoring this, although there is a high degree ofcorrelation between the copy number of CCL3L andCCL4L genes, individuals contain more copies ofCCL3L than CCL4L [32]. We therefore tested thehypothesis that the overall phenotypic impact of thechemokine gene-rich locus at chromosome 17q12 regionon HIV-AIDS susceptibility will be influenced by thecombinatorial content of different CCL3L and CCL4L-containing segmental duplications. To test this, wedeveloped specific probes for the distinct CCL3L andCCL4L genes and determined their associations withHIV-AIDS susceptibility in a cohort of children exposedperinatally to HIV-1 from the Ukraine.

Methods

CohortWe studied a cohort of 298 children from Ukrainewho were exposed perinatally to HIV-1 between 1998and 2006. Of these, 178 (59.7%) were infected. Thisdistribution is not indicative of the perinataltransmission rate for HIV-1 infection, as the recruitmentof children was based on HIV status. DNA was alsoavailable from the mothers of 89 children. To minimizepotential confounding due to population stratification,only children and mothers of Slavic descent, the mostcommon ethnic group found in Ukraine, were includedin the study. These patients were recruited at the mainoutpatient HIV-1 reference centers (‘Municipal Centersfor HIV Management and Prophylaxis’) in the Dnepro-petrovsk region. All pregnant women within this regionwho test positive for HIV are referred to these centers.Additionally, all HIV-positive children born to thesemothers also receive their medical care in these centers.Informed written consent was obtained from parentsor legal guardians of all the children, and directly fromthe adults. The study was approved by the InstitutionalReview Boards of the participating institutions.The characteristics of the study individuals are givenin Table 1.

Study outcomesThe main outcome measures were HIV serostatus andprogression to AIDS [1993 criteria of the Center forDisease Control and Prevention (CDC) classification forchildren].

GenotypingTaq Man real-time assays similar to those describedpreviously [24] and described in the Supplementary

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CCL3L and CCL4L in HIV-AIDS pathogenesis Shostakovich-Koretskaya et al. 681

Table 1. Characteristics of the children included in the study.

Parameter HIV infected HIV uninfected P

Number of children 178 (59.7%) 120 (40.3%) –Did not receive ZDV prophylaxis 123 (69.1%) 44 (36.7%) 3.2�10�8

Received breastfeeding [n (%)] 17 (9.6%) 5 (4.1%) 0.077Hemoglobin (g/dl) [mean (SE)] 10.8 (0.09) 11.1 (0.11) 0.030RBC count [�106/ml; mean (SE)] 4.21 (0.08) 4.36 (0.29) 0.264Baseline CD4þ T-cell count (cells/ml) [mean (SE)] 968.2 (62.2) 2515.8 (11.2.9) <1�10�22

Baseline CD4 percentage [mean (SE)] 22.9 (0.82) 38.8 (0.75) <1�10�22

Average age at initiation of HAART [years, mean (SE)] 5.00 (0.35) – –Children developing AIDS [n (%)] 56 (31.5%) – –Total duration of follow-up (person years) 735.33 – –Duration of follow-up [years, median (IQR)] 7.63 (5.29) – –

ZDV, zidovudine; P, significance value; SE, standard error.

Materials and Fig. S1 were used to quantify the CNV ofCCL3L and CCL4L genes. Primer and probe sequencesare listed in Supplementary Table 1.

Statistical analysisThe association between gene copy number and risk ofacquiring HIV infection was assessed by using logisticregression analyses, and rate of disease progression wasinvestigated by using Kaplan–Meier survival curves andCox proportional hazards modeling. Attributable frac-tion was estimated as described previously [24]. Thepattern of correlations among the copy numbers of thedifferent CCL3L and CCL4L genes was determinedusing principal components analyses. For this, a factorsolution provided by using a minimum eigenvalue ofone with the results optimized using the varimaxrotation was used. Details of factor analyses are asdescribed in the Supplementary Material. All statisticalanalyses were conducted using the Stata 7.0 (StataCorporation, College Station, Texas, USA) softwarepackage.

Results

Nomenclature and PCR assay precisionIn this study, we denoted CCL3L1 (MIM:601395),CCL3L2 (MIM:609467), CCL3L3 (MIM:609468),CCL4L2 (MIM:610757) and CCL4L1 (MIM:603782)as CCL3La, CCL3Lb, CCL3La, CCL4La and CCL4Lb,respectively (Fig. 1a and b), for the following reasons. Inprior studies [24–32], because of the nature of the PCRprimer probes used, we and others assessed the copynumber of all CCL3L genes (CCL3L1, CCL3L2,CCL3L3) and had previously designated this compositeas the CCL3L1-containing segmental duplication (Fig. 1aand b). CCL3L1 and CCL3L3 are separate genes, eachhaving three identical exons that encode identicalproteins [3,5], and therefore they are together denotedhere as CCL3La (CCL3L1þCCL3L3¼CCL3La;Fig. 1a–c). On the basis of current literature, CCL3L2,designated here as CCL3Lb, is thought to contain only

opyright © Lippincott Williams & Wilkins. Unauth

two exons whose sequences are identical to those foundin exons 2 and 3 in CCL3L1 and CCL3L3 [3,5]. BecauseCCL3Lb (CCL3L2) lacked the first exon found inCCL3La (CCL3L1 or CCL3L3), it has been consideredas a pseudogene [3,5]. However, by bioinformatics andmRNA profiling, we identified novel 50 exons forCCL3Lb, which give rise to two alternatively splicedtranscripts (CCL3Lb-v1 and CCL3Lb-v2, Fig. 1c anddata not shown). These alternatively transcribed mRNAspecies contain chemokine-like domains but are notpredicted to encode classical chemokines (data notshown). Hence, CCL3La and CCL3Lb transcribe twodistinct kinds of transcripts; the former encoding classicalchemokines and the latter encoding alternatively splicedtranscripts with novel 50 exons (Fig. 1c).

Similarly, CCL4L represents a composite of CCL4Lb(CCL4L1) and CCL4La (CCL4L2), two distinct genesthat have identical exonic sequences (Fig. 1a and b, [3,5]).Although these two genes have identical exonicsequences, a (A!G) transition in the splice acceptorsite in the intron II of CCL4Lb relative to CCL4La leadsto generation of aberrantly spliced CCL4Lb transcripts,that is, formation of transcripts with retention of portionsof intron II or partial loss of exon 3 (Fig. 1d). Hence,CCL4La produces transcripts that are predicted toencode a classical chemokine, whereas CCL4Lb trans-cribes aberrantly spliced mRNA species (Fig. 1d).

On the basis of the aforementioned, we developed twoseparate assays to quantify the total copy number of allCCL3L or CCL4L genes, and separate assays each forthe individual components of CCL3L (CCL3La andCCL3Lb) and CCL4L (CCL4La and CCL4Lb). ThePCR assays we developed estimated the copy number ofthese individual chemokine gene CNVs with highprecision (Fig. S1). The estimates of the sum of thecopy number of CCL3La and CCL3Lb or CCL4La andCCL4Lb using these individual PCR assays were verysimilar to the estimate of the total copy number ofCCL3L or CCL4L genes, respectively, with 100%concordance between the estimates for zero, one, twoor at least two copies (data not shown).

orized reproduction of this article is prohibited.

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682 AIDS 2009, Vol 23 No 6

Segmental duplicated region 17 q12

Telomere

CCL3La

CCL4La

CCL4Lb

CCL3Lb_v1CCL3Lb_v2

CCL3La

CCL3Lb

CCL4La

CCL4Lb

Variants

14k 14k 14k12k70k89k

1 32

1 32 II

III

I

1 32

1b1a 32II IIII

III

gt ag ag ag ... ag ...

gt ag ag gg ... ag ...

21 3 4 5 Total07

7811 203

17 28251 7

911 2

93445

214787

1

0123456

Total 7 89 220 54 17 2 389

CCL4L

CC

L3L

Nomenclatureused NCBI (Gene ID)

CCL3L1 (6349)

CCL3L3 (414062)

CCL3L2 (390788)

CCL4L2 (388372)

CCL4L1 (9560)

CCL3La

CCL3Lb

CCL4La

CCL4Lb

CCL3L

CCL3CCL4

(CCL3

L1)

(CCL4

L1)

(CCL3

L2)

(CCL3

L3)

(CCL4

L2)

CCL4La

CCL3La

CCL3Lb

CCL4Lb

CCL3La

CCL4L

CCL3L copy number

CC

L4L

copy

num

ber

0

5

4

3

2

1

0

2 4 6

Pearson’s r = 0.92

(a)

(b)

(e) (f)

(c)

(d)

Fig. 1. Chromosome 17q12 segmental duplication, chemokine gene copy number variation nomenclature and correlationbetween copy number of CCL3L and CCL4L. (a) Schematic representation of CCL3, CCL4, CCL3L and CCL4L genes. Arrowsindicate the orientation of each gene. Shown on top is the distance between the indicated genes. Map is not to scale. (b)Nomenclature of CCL3L and CCL4L genes used here and previously [9,24,26,28,30,31]. (c) and (d) Schematic representation of theexon–intron structure of (c) CCL3L and (d) CCL4L genes. In (d), the downward pointing arrow indicates the A!G transition thatleads to the generation of aberrantly spliced CCL4Lb transcripts, and the splicing patterns of these mRNA species are indicated bythe curved arrows. Thus, a CCL4L copy, designated here as CCL4La, has the intact AG intron–exon splice sequence and ispredicted to transcribe an intact full-length CCL4L transcript. By contrast, the other CCL4L copy, designated here as CCL4Lb, has amutated intron–exon splice site sequence (GG), and this results in the formation of a set of aberrantly spliced transcripts that arepredicted to produce CCL4L proteins that may not function as classical chemokines (as described by Colobran et al. [9]). Boxesrepresent exons, which are identified by Arabic numerals and the lines joining the boxes are the introns, which are identified byRoman numerals. Figure not to scale. (e) Scatter plot showing the correlation of the CCL3L and CCL4L genes copy numbers for allstudy individuals. (f) Distribution of CCL3L and CCL4L gene copy number among all study individuals. Grey represents number ofindividuals who have the same dose of CCL3L and CCL4L. The numbers below the grey boxes represent the number of individualsthat have more copies of CCL3L than CCL4L.

CCL3L–CCL4L dose and HIV-AIDS riskThe distribution of CCL3L copy number in UkrainianHIV-positive or HIV-negative children and HIV-positivemothers were similar to those observed in individuals ofEuropean descent [24], and the average copy number ofCCL3L in this population was two (Fig. S2a). Althoughthe copy number of CCL3L and CCL4L were highly


correlated (92%, P< 0.0001), the gene dose of CCL3Lwas higher than that for CCL4L (Fig. 1e and f). Given thishigh correlation, predictably, the distribution of CCL4Land CCL3L were very similar (compare Fig. S2a and b),and therefore associations of the total CCL4L gene dosewith HIV-AIDS susceptibility would be expected to besimilar to those detected for the total CCL3L gene dose.


C


100 4.00

3.50

Odd

s ra

tio

Odd

s ra

tio

Per

cent

age

Per

cent

age 3.00

2.50

2.00

1.50

1.00

0.50

0.00

80

60

40

20

0

100 4.00

3.50

3.00

2.50

2.00

1.50

1.00

0.50

0.00

80

60

40

20

0

>2 >2

>2

>2

<2

<2 <2

<2

>2

>2

>2 >2

<2<2<2<2

HIV+n = 178

HIV--n = 120

HIV+n = 178

HIV--n = 120

Una

djus

ted

Adju

sted

Una

djus

ted

Adju

sted

P =0.071

P =0.074

P =0.075

P =0.072

55

10

13

11

1.00

1.04

1.21

10.4

---

0.96 -- 1.12

0.53 -- 1.33

4.39 -- 14.5

1.00

1.84

1.30

12.9

---

0.42

0.70

0.02

---

0.42 -- 8.08

0.34 -- 5.01

1.41 -- 118.2

---

0.34

0.57

9.3x10--8

0 20 40 60 80 100

Copy number

Child Mother N

% of HIV-infectedchildren Unadjusted analysis Adjusted for prophylaxis

OR 95% Cl ORP P95% Cl

CCL4L copy number and risk of HIV acquistionCCL3L copy number and risk of HIV acquistion(a) (b)

(c)Risk of HIV transmission based on the CCL3L-CCL4L copy number in mother-child pairs

Fig. 2. Association of the CCL3L and CCL4L gene copy number with risk of acquiring HIV infection. (a and b) The risk of HIVacquisition based on the (a) CCL3L and (b) CCL4L copy number in children. Left panel shows the distribution of CCL3L or CCL4Lcopy number categorized as less than two or at least two in the HIV-positive and HIV-negative children. Right panel shows resultsof logistic regression analyses unadjusted (shown in green) and adjusted (shown in red) for receipt of ZDV prophylaxis. Thediamonds and error bars represent the OR and 95% CI, respectively. (c) Association of CCL3L copy number in the mother–childpairs with the risk of vertical transmission. The data on the left shows the distribution of the 89 mother–child pairs by gene copynumber and the bar chart shows the proportion of children within each of the four categories who acquired HIV infection. The riskof HIV acquisition was estimated using multinomial logistic regression analysis which was unadjusted (left) and adjusted (right) forreceipt of ZDV prophylaxis. ZDV, zidovudine; CI, confidence interval; OR, odds ratio; P, significance value.

In a previous study [24], we found that in European–Americans or Argentinean children, possession of acopy number of CCL3L that was less than the averagefound in the overall population (two) was associatedwith an increased risk of acquiring HIV infection.Concordantly, among Ukrainian children exposedperinatally to HIV, possession of a copy number ofCCL3L or CCL4L that was less than two was associatedwith a trend for a nearly 75% increased risk ofacquiring HIV before or after accounting for receipt ofzidovudine (ZDV) prophylaxis, respectively, comparedwith children who had at least two copies of CCL3L orCCL4L (Fig. 2a and b).

We next determined whether risk of transmission differedbased on whether a mother and her offspring had similaror dissimilar doses of CCL3L. Compared with mother–child pairs in which both the mother and her offspringhad at least two CCL3L copies, the risk of transmissionwas significantly higher by a factor of approximately 10–13 only when both the mother and her offspring had aCCL3L gene dose of less than two (Fig. 2c). In these 89


mother–child pairs, identical associations were detectedfor the copy number of CCL4L. HIV-positive childrenwith less than two CCL3L or CCL4L copies hadexperienced an approximately two times faster rate ofprogression to AIDS than those with a higher gene dose(Fig. 3a and b).

Relationships among CCL3L and CCL4L genesWe next investigated the phenotypic impact of thedifferent components of CCL3L and CCL4L genes.Figure S2 (c) and (d) show the distribution of the copynumbers of the different CCL3L and CCL4L genes inthe study individuals. The distribution patterns ofCCL3La and CCL4La were very similar to thoseof the total copy numbers of CCL3L and CCL4L,respectively, whereas most study individuals lackedCCL3Lb and CCL4Lb (Fig. S2).

The pair-wise comparisons of the copy number ofthe different CCL3L and CCL4L genes revealedthe following pattern. There was a highly statisticallysignificant positive correlation between the copy numbers


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684 AIDS 2009, Vol 23 No 6

1.00

(a)

0.75

0.50

0.25

0.000 5 10

Time (years)0 5 10

Time (years)

0 2 4 6 8 10Time (years)

Pro

port

ion

AID

S-f

ree

1.00

(b)

0.75

0.50

0.25

0.00

Pro

port

ion

AID

S-f

ree

1.00

(d)(c)

0.75

0.50

0.25

0.00Pro

port

ion

AID

S-f

ree

RH1.99

95% Cl0.98 -- 4.09

P0.058

RH2.11

RH1.001.844.08

95% Cl1.04 -- 4.25

95% Cl

0.88 -- 3.851.43 -- 11.64

P

0.1040.009

N118236

P0.036

<2 CCL3L copies<2 CCL4L copies

CCL4La>0

>00

0CCL4Lb

≥2 CCL3L copies ≥2 CCL4L copies

1 1

CCL3La

CCL3Lb

CCL4Lar = 0.44r = 0.36

00

1

20

50

1

2

2 4 56Copy number

Cop

y nu

mbe

r

0 01 2

r = 0.75 r = 0.30

r = 0.56

r = --0.30

CC

L3Lb

CC

L4La

CC

L4Lb

Fig. 3. Influence of the CCL3L copy number on HIV disease progression in children who did not receive perinatal zidovudineprophylaxis. (a) Kaplan–Meier plots for time to AIDS stratified by CCL3L copy number in Ukrainian HIV-1-infected children(n¼123). Pink curve is for HIV-positive children with at least two CCL3L copy number, whereas green curve is for individuals withless than two copies of CCL3L. (b) Same as in (a), data are for CCL4L copy number. (c) Matrix of correlations among the indicatedCCL3L and CCL4L genes in all study participants. The pair-wise comparisons revealed a highly statistically significant positivecorrelation (P<0.001 for each pair-wise comparison) except for the frequencies of CCL4La and CCL4Lb, which displayed aninverse correlation. (d) Kaplan–Meier plots of subgroups based on CCL4La and CCL4Lb copy number and their effects on diseaseprogression in infected children (n¼147). Zero (0) indicates that the gene is not present, 1 indicates the presence of one copynumber of CCL4La or CCL4Lb. The red-colored Kaplan–Meier plot is for individuals who lack CCL4La, but have one or morecopies of CCL4Lb; the green Kaplan–Meier plot is for individuals who have one copy each of CCL4La and CCL4Lb; and theblue Kaplan–Meier plot is for those who lack CCL4Lb and have one or more copies of CCL4La. The latter group representsthe reference category. CI, confidence interval; N, number of children; P, significance value; r, Spearman’s correlation coefficient;RH, relative hazard.

of CCL3La and CCL3Lb, CCL3La and CCL4La,CCL3La and CCL4Lb, CCL3Lb and CCL4La(Fig. 3c). In contrast, there was a highly statisticallysignificant negative correlation between the copy numberof CCL4La and CCL4Lb (r¼�0.30, P< 0.0001; Fig. 3c).

On the basis of the observed inverse relationship betweenCCL4La and CCL4Lb (Fig. 3c) and because CCL4La butnot CCL4Lb-derived transcripts are predicted to encodea classical chemokine (Fig. 1d), we surmised thefollowing: those who had more ‘functional’ chemo-kine-encoding CCL4La copies than CCL4Lb copiesmight fare better in terms of their disease course thanthose who only possessed CCL4Lb copies. To test thispossibility, we stratified HIV-infected children into three


groups as shown in Fig. 3d. Consistent with ourhypothesis, possession of only CCL4Lb was associatedwith a four-fold faster rate of disease progression thanthose who only possessed CCL4La (compare blue and redKaplan–Meier plots, Fig. 3d). By contrast, those who hadone copy of CCL4La and CCL4Lb displayed anintermediate disease phenotype (green Kaplan–Meierplot, Fig. 3d). The Kaplan–Meier plot of individuals whohad higher copy numbers of CCL4La than CCL4Lb wassimilar to those who only possessed CCL4La (data notshown). These findings underscored the beneficial andnegative associations of CCL4La and CCL4Lb copies onHIV disease course, respectively. There were too fewindividuals who were null for CCL3La to conductsimilar analyses.


C


--1

--1

1

4

3

2

1

0

0.0

0.2

0.4

0.6

Odd

s ra

tio/r

elat

ive

haza

rd

Fac

tor

scor

e ra

tio (

f1/f2

)

0.8

1.0

1.2 Risk ofinfection

Rate ofprogression

1

Factor 1

Fac

tor

2

Factor loadings

(a) (b) (c)

CCL3Lb

CCL3La

CCL4La

CCL4Lb

HIV-- HIV+ HIV+

Children Mothers

P = 0.001

P = 0.001

P = 0.042

Una

djus

ted

Una

djus

ted

Adju

sted

Adju

sted

P = 0.058P = 0.013P = 0.025

Fig. 4. Factor analysis in study individuals. (a) Results of principal components analyses. Two orthogonal factors (represented byabscissa and ordinate) were retained on the basis of an eigenvalue of more than one. The plot represents the varimax-rotatedloadings for each of the components on the two factors (factors 1 and 2). Considering these loading patterns, the first factorrepresents mainly CCL3La and CCL4La, the two genes that transcribe functional chemokines, whereas factor 2 represents mainlyCCL4Lb, which transcribes aberrantly spliced mRNA species. (b) Distribution of the factor 1/factor 2 (f1/f2) score ratio in studygroups. HIV-positive children as well as their mothers had a significantly lower f1/f2 score ratio as compared with the HIV-negativechildren as assessed by Mann–Whitney test. For these analyses, we took the ratio of the scores for the first and second factors for agiven study individual. As the factor scores were scattered over the interval (�2.21 and 4.27), we linearly transformed the rawfactor scores by adding a constant of 2.5. This transformation converted all the factor scores into positive real numbers withoutaffecting the original distribution of the factor scores. We then took the ratio of these positive factor scores. (c) Association of thelog-transformed f1/f2 ratio with the risk of acquiring HIV (analyzed using logistic regression) and rate of disease progression(analyzed using Cox proportional hazards regression). The diamonds and error bars represent the point estimates and 95% CIs forOR (for risk of HIV acquisition) and relative hazards (for rate of disease progression). Results are from unadjusted and adjusted (forthe receipt of prophylaxis) regression models. The numbers at the bottom show the significance values. CI, confidence interval;OR, odds ratio.

CCL3L–CCL4L components and HIV-AIDSsusceptibilityThe importance of accounting for the complexityimposed by the combinatorial gene content generatedby variable CNV on the 17q12 was underscored by theobservation that there was a positive correlation betweenthe frequencies of each of the CCL3L and CCL4L genesexcept for the pair-wise comparison of CCL4La andCCL4Lb (Fig. 3c) and the contrasting disease-influencingeffects associated with these two genes (Fig. 3d). Thisraised the possibility that the relative balance between thecontent of ‘protective’ versus ‘detrimental’ duplicatedchemokine genes influences the HIV-AIDS-influencingphenotypic effect of the chemokine gene-rich 17q12regions that have undergone segmental duplications. Weused principal components analyses to address thispossibility as well as to gain a greater understanding ofthe observed pattern of correlations between thechemokine gene copy numbers.

By factor analyses, we found that these four componentsloaded differentially onto a two-factor solution (Fig. 4a):CCL3La and CCL4La loaded heavily onto the first factor(factor 1), whereas the second factor (factor 2) wasrepresented by CCL4Lb loading very highly onto it. Ofnote, CCL3Lb loaded almost equivocally on both the


factors suggesting that this is a distinct component. Thedegree of uniqueness of CCL3La, CCL3Lb, CCL4Laand CCL4Lb was 0.05, 0.59, 0.04 and 0.11, furtheremphasizing that with regards to the copy numberdistribution, the copy number of CCL3Lb was uniquelydifferent from the distribution of CCL3La, CCL4La andCCL4Lb. Thus, factor 1 largely reflects CCL3La andCCL4La, whereas factor 2 largely reflects CCL4Lb.

To assess whether biological interpretations made fromthe correlation patterns and principal components analyseswere justifiable, we conducted the following analyses.Using the results from principal components analyses, wegenerated standardized scores for the first and second factorfor each individual. On the basis of this, we surmised that alower score for factor 1, which equates with a lowernumber of CCL3La and CCL4La copies would morelikely associate with an HIV-positive status. Consistentwith this, we observed that themedian for the factor 1 score(in parenthesis) in HIV-negative children (0.25) wassignificantly higher than that found in HIV-positivechildren (0.13; P¼ 0.042 for comparison) and HIV-positive mothers (�0.43, P¼ 0.0067 for comparison).These observations suggested that a reduction in theoverall gene content of CCL3La and CCL4La confers adifferential risk of acquiring HIV.


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We next examined whether the relative content of thesetwo factors in a given individual was a determinant of therisk of acquiring HIV infection and progressing to AIDS.A ratio of the factors 1 (f1) and 2 (f2) scores (referred tohere as f1/f2 score ratio) exceeding one indicates a relativeenrichment of the first factor, whereas a value less thanone indicates a relative enrichment of the second factor.We found that HIV-uninfected children had significantlyhigher values of the f1/f2 ratio compared with HIV-infected children and HIV-infected mothers (Fig. 4b).Using logistic regression analysis, we then determined ifthe risk of HIVacquisition is influenced by the f1/f2 scoreratio. We found that each log-fold increase in the f1/f2score ratio was associated with approximately 43 and 41%reduced likelihood of acquiring HIV before and afteraccounting for ZDV prophylaxis, respectively (Fig. 4c).Similarly and to a comparable extent, we found by usingCox proportional hazards regression analyses that a log-fold increase in the f1/f2 score ratio was associated with anapproximately 38 and 37% decreased rate of progressionto AIDS in the HIV-infected children before and afteraccounting for the receipt of prophylaxis, respectively(Fig. 4c).

Discussion

The present study has two major findings. First, theyconfirm and amplify the results of previous studies whichshowed that a low dose of CCL3L genes (referredpreviously as the CCL3L1-contianing segmental dupli-cation) is associated with an increased risk of acquiringHIV and progressing rapidly to AIDS. Furthermore, ourresults demonstrate that a low CCL4L gene dose hassimilar associations. These results add credence to thenotion that these genetic determinants may influenceepidemic spread of HIV. Successful transmission of HIV-1depends on both the infectiousness of the HIV-positiveindividual and susceptibility of the HIV-negative indi-vidual. Infectiousness is in part dependent on the viralload [37,38], and previous studies [24,26,29–31] indicatethat a low copy number of CCL3L is associated with aprogressive HIV disease course and higher viral load.Thus, it follows that mother–child dyads in which bothmembers have a low copy number of CCL3L should havethe highest probability of transmission, which is what weobserved in this study. In 12.4% of mother–child pairs,both the mother and the child possessed less than twoCCL3L (or CCL4L) copies and 91% of these childrenhad acquired HIV infection. This translated to an oddsratio of more than 10 for the risk of transmission, which isamong the highest odds for transmission that we haveobserved, and an attributable fraction of 18.4% (95%confidence interval 6.1–30.6%). These results suggestthat a substantial proportion of vertical transmission in ourstudy individuals may be explained by mother–childdyads in which each member has a low CCL3L/CCL4L


dose. By analogy, we hypothesize that a similarly highproportion of transmission may occur among HIV-positive/HIV-negative sexual partner pairs when eachmember has a low CCL3L dose, a premise that is alsosupported by our modeling studies [39]. As there wascomplete concordance in the gene copy numbers ofCCL3L and CCL4L in the mother–child pairs in whichboth mother and offspring had less than two CCL3Lcopies, these data also reflect the effects of the overallCCL4L gene copy number on transmission.

Second, our findings illustrate a chromosomal regionwhose genomic architecture is highly complex such thatindividuals do not inherit the same complement ofCCL3L and CCL4L genes. However, we suggest thatuncovering this complexity might be necessary to definethe true phenotypic impact of these duplicated genes onHIV-AIDS susceptibility. This is exemplified by thefinding that the copy numbers of CCL4La and CCL4Lbare inversely correlated and have opposing effects onAIDS progression rates, despite the fact that sequences ofthe coding exons of these two genes are identical [3,5].Furthermore, we show that the balance between the copynumbers of the genes that transcribe classical versusaberrantly spliced CCL3L and CCL4L mRNA speciesinfluences HIV-AIDS susceptibility; a higher genecontent of CCL4Lb (CCL4L1) or a lower content ofCCL3La and CCL4La increased the risk of transmissionand an accelerated disease course. Underscoring thenegative influence of CCL4Lb on HIV acquisition, aprevious study [9] also found that HIV-positive individ-uals were enriched for more copies of this gene comparedwith HIV-negative controls.

It is conceivable that there are additional tiers of geneticand mRNA complexity in this locus. We have identifiednovel 50 exons for CCL3Lb, a gene that was previouslythought to only have two exons, which are homo-logous to exons 2 and 3 of CCL3L1 or CCL3L3; for thisreason, CCL3Lb was considered as a pseudogene [5]. Wefound that these novel 50 exons give rise to twoalternatively (CCL3Lb-v1 and CCL3Lb-v2) splicedtranscripts. These transcripts contain chemokine-likedomains, but are not predicted to encode classicalchemokines, and additional studies will be required touncover the functional properties of these CCL3LmRNA species.

The present findings have four implications. First,although CCL4La and CCL4Lb share 100% sequenceidentity in their coding sequences, the findings presentedherein and those from our preliminary studies in otherpopulations indicate that the pair-wise correlationsamong and between CCL3L and CCL4L genes, andthe degree of the inverse correlation between the copynumber of CCL4La and CCL4Lb genes may varybetween populations. This suggests that genotype–phenotype association studies may need to account for


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interpopulation differences in the genomic architectureof this locus.

The second implication of our results is that theassessment of CCL4L dose is capturing the sum of twogenes (CCL4La and CCL4Lb) whose copy numberfrequencies are inversely related and who have opposingeffects on HIV disease course. Thus, the true phenotypicimpact of CCL4La and CCL4Lb cannot be made usingCCL3L copy number as a proxy for CCL4L or byevaluation of the composite CCL4L. This might explainin part why previous studies may not have found anassociation between CCL4L copy number and HIVdisease [40]. Third, the CCL3Lb copy number loadedweakly and equivocally on factor 1 or 2, indicating thatthis gene may influence HIV-AIDS pathogenesis by as yetunidentified means. Consistent with this possibility, wehave found that CCL3Lb (CCL3L2) generates alter-natively transcribed transcripts whose 50 exons differ fromthose of CCL3La (CCL3L1 and CCL3L3) (Fig. 1c).Fourth, greater insights into the immune correlates linkedto a high gene dose of specific CCL3L and CCL4Lchemokine genes that confer protective effects mayprovide novel insights into the correlates of protection.The importance of elucidating these correlates is alsohighlighted by the recent observation that CCL3L copynumber may influence susceptibility to Simian-AIDS inrhesus macaques [34,41].

In summary, we confirm and extend significantly priorresults indicating that a low copy number of CCL3Linfluences risk of HIVacquisition and disease progressionin a cohort of children exposed perinatally to HIV. Wedemonstrate a role for CCL4L CNV in HIV-AIDSsusceptibility. However, we show that dissecting thecombinatorial genomic complexity posed by varyingproportions of distinct CCL3L and CCL4L genes amongindividuals is required to elucidate the completephenotypic impact of this locus in HIV, and suggest thatthe balance between the copy number of the differentCCL3L and CCL4L genes influences HIV-AIDSsusceptibility. From a broader perspective of relevanceto the CNV field, it might be important to determinewhether the combinatorial genomic content generatedby distinct genes within a copy number variable regiondetermines disease susceptibility.

Acknowledgements

Supported by award no. UKB2-2705-DP-05 of the U.S.Civilian Research and Development (CRDF) to L.S.K.and S.K.A., the Veterans Administration Center on AIDSand HIV infection at the South Texas Veterans HealthcareSystem and a MERITaward (R37046326) from the NIHto S.K.A. This work was also supported by a Elizabeth


Glaser Scientist Award and Burroughs Wellcome ClinicalScientist Award in Translational Research to S.K.A.

We thank George Crawford for invaluable programmaticsupport at UTHSCSA; Vince Marconi for critical advice;staff members and patients at the Department of Pediatricsat Dnepropetrovsk State Medical Academy for logisticsupport and participation in the study, respectively; BirjuShah, Vivian Ahn, Lisa Beachy and Jill King for technicalassistance; and A.S. Ahuja for forbearance. Any opinions,findings, conclusions or recommendations expressed inthis material are those of the authors and do not reflect theviews of CRDF.

L.S.K. and S.K.A. conceived the project, obtainedfunding for the study and provided expertise intodifferent aspects of the study. L.S.K. and Z.A.C. enrolledpatients, collected samples, conducted experiments andanalyzed data. G.C. and W.H. designed the experimentalprotocols and analyzed data. R.S. helped establishing thePCR assays. G.G., S.M. and S.K.A. planned andperformed the mRNA cloning and sequencing work.M.J.D., S.S.A. and R.A.C. provided critical conceptualinput and analyzed data. S.K.A., H.K. and G.C. wrote theinitial draft and all authors critically reviewed themanuscript and provided feedback. H.K. and S.K.A.conceived the statistical design.

There are no conflicts of interest.

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Genotyping methods

Nomenclature of CCL3L and CCL4L chemokine genesis as illustrated and described in the main text (Fig. 1a,b).DNA was isolated from EDTA-treated whole blood byusing a Qiagen kit. The copy number of CCL3L(previously designated as the CCL3L1-containing seg-mental duplication and includes CCL3L1/2/3) wasgenotyped as described previously [1]. Similar Taqmanbased Real-time PCR assays were developed to genotypethe copy number of the other chemokine genes. Briefly,the human housekeeping beta-globin (BGB) gene was usedas an internal control as a gene with two copies per diploidgenome (pdg). The A431 cell line was used as a standardfor CCL3La, CCL4L and CCL4La with each genepossessing two copies pdg ([2] and data not shown). TheK562 cell line was used as a standard for CCL3Lb andCCL4Lb with one and two copies pdg, respectively (datanot shown). CCL4La and CCL4Lb were run in a duplexassay whereas the others were run in single-plex. Eachsample was run in triplicate in three separate 384-wellplates. Rounded average numbers were used for analysisand quality control procedure was applied as describedpreviously [1]. Primer and probe sequences are listed inthe table below.

Statistical methods

In this study, we examined the association of the overallCCL3L and CCL4L gene copy number and theircomponents (CCL3L1a, CCL3L1b, CCL4La, andCCL4Lb) with two end-points: risk of HIV acquisitionand rate of progression to AIDS. To summarize thecorrelation patterns across the components of the CCL3Land CCL4L genes we conducted factor analysis using themethod of principal components. For this, we firstextracted significant factors (defined as factors with aneigenvalue exceeding unity) and then applying a varimaxrotation to the extracted orthogonal factors. In the nextstep, we generated factor scores for each extracted factorfor each individual based on the results obtained from theprevious step. We then determined the association ofthese factor scores with risk of infection and rate ofprogression. Greater details of the method of factoranalysis are discussed below.

Factor analysisFactor analysis provides an unbiased exploratory means tounderstand complex correlation patterns among severalvariables and to distill these complex patterns into aparsimonious mathematical solution called as factors.Highly correlated variables load onto a common factorand highly uncorrelated variables segregate onto differentfactors. We used a method of principal components toparse out the complex correlation pattern among thecomponents of the duplicated genes on chromosome


17q12, i.e., CCL3La, CCL3Lb, CCL4La and CCL4Lb(shown in Fig. 4a). For deriving the factor solution weused a criterion of a minimum eigenvalue of 1.

Using this criterion we arrived at the following factorsolution:

These results show that there were only two retainedfactors based on the four variables (genes) – in otherwords the correlation pattern of the components of theduplicated segment could be summarized using twofactors. Since principal components analysis assumes thatthe factors are orthogonal (i.e., the factors are themselvesuncorrelated), we next used the varimax rotation to arriveat an optimized factor solution as follows:

These results are depicted in Fig. 4a and suggest that theCCL3La and the CCL4La gene copy numbers loadedheavily on the first factor while the CCL4Lb gene copynumber loaded onto the second factor. The CCL4Lagene copy number also strongly negatively loaded ontothe second factor. Interestingly, the CCL3Lb gene copynumber loaded equivocally and moderately onto boththe factors.

In the table of results from rotated factor loadings (seeabove), the uniqueness of each variable is a measure of the


Copyright © Lippincott Williams & Wilkins. Unauthor

690 AIDS 2009, Vol 23 No 6

Fig. S1. Distribution of copy number of the CCL3L andCCL4L gene components, i.e., CCL3La, CCL3Lb, CCL4Laand CCL4Lb among study subjects. Each dot represents thecopy number of the indicated gene in the study subjects.Individual subjects are distributed along the abscissa, whilethe persons estimated copy number of the indicated genes isdepicted along the ordinate. Note, the copy number for eachindividual cluster around the integers (e.g., 0, 1, 2 etc),demonstrating the precision of the PCR assay.

Fig. S2. Distribution of the CCL3L and CCL4L gene copy number acopy number among the indicated study subjects. (c-d) DistributionCCL4L (d) genes in the indicated study groups.

degree of correlation of the indicated gene copy numberswith the factors that were mathematically extracted. It canbe seen that the uniqueness of CCL3La, CCL4La andCCL4Lb is low indicating that these gene copy numbersare strongly correlated with the two factors. However, theCCL3Lb is highly unique and only minimally loads ontoeither factor. Therefore, it can be surmised that theCCL3Lb gene copy number provides unique informationthat is distinct from that provided by the other threevariables.

In the next step, to translate the results of factor analysisinto clinically and genetically meaningful information,we generated factor scores for each individual. In theory,since the variables load onto factors it is possible toestimate the degree to which an individual might loadonto that factor given that individual’s data on thecontributing variables. For example, let us assume that thestandardized (z-value/score) results of the Taqman PCRassays for the four components were 1.5, 0.04, 1.2, and�0.5 for CCL3La, CCL3Lb, CCL4La, and CCL4Lb,respectively, then that individual’s score for the first factorwill be (based on the coefficients shown in the tableabove): (0.89� 1.5)þ (0.44� 0.04)þ (0.94� 1.2)þ(�0.03��0.5)¼ 2.50. The same individual’s score onthe second factor will be (0.39� 1.5)þ (0.47� 0.04)


mong study subjects. Distribution of (a) CCL3L and (b) CCL4Lof the copy numbers of each component of the CCL3L (c) and

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Supplementary Table 1. Primers and probes used for assays to quantify the gene copy number of CCL3L and CCL4L genes. CCL3L was quantifiedas described previously [1].

Gene Primers/Probes Oligonucleotide sequences

CCL3La Sense primer 50-GGGTATGACTTCTTGAACCGACAAA-30

Antisense primer 50-GGTTCTCTGTTTCTCTATGTGATCCA-30

Probe 6FAM-CATGAAGAGAGCTAAGAGAAC-MGBNFQCCL3Lb Sense primer 50-CATCCACTCGCTCACACCTGTA-30

Antisense primer 50-GCGGTCGGCGTGTCA-30

Probe 6FAM-AGAGTTGGGCTTATTCT-MGBNFQCCL4L Sense primer 50-CATGGTCAGGCAGAGGAAGATG-30

Antisense primer 50-GCTTGCCTCTTTTGGTTTGGAAT-30

Probe 6FAM-TACCACAGGCAAGGGAT-MGBNFQCCL4La and CCL4Lb Sense primer 50-GGAAGATGCCTACCACAGGC-30

Antisense primer 50-GCGCAGACTTGCTTGCC-30

CCL4La Probe VIC-CTTGTTCTACaGATTCC-MGBNFQa

CCL4Lb Probe 6FAM-CTTGTTCTACgGATTCC-MGBNFQ a

BGBb Sense primer 50-TCGCTTTCTTGCTGTCCAATTTCTA-30

Antisense primer 50-ATGCTCAAGGCCCTTCATAATATCC-30

Probe VIC-CCTAAGTCCAACTACTAAACTG-MGBNFQ

VIC and FAM, probe reporter fluorescent dyes. MGBNFQ, Molecular-Groove Binding Non-fluorescence Quencher hybridization probes whichallow for using probes at lower melting temperature.aProbe sequences in lower case represent difference between CCL4La and CCL4Lb.bBGB, Human housekeeping beta-globin (BGB) gene.

þ (�0.26� 1.2)þ (0.94��0.5)¼�0.18. Thus, thisindividual will have a high score for the first factor anda negative score for the second factor.

Having generated the scores for each individual, weproceeded to study the association of the factor scores wecreated a composite factor score ratio by taking the ratioof the first factor score and second factor score.Conceptually, this ratio represents the expansion orshrinkage of the first factor score relative to the secondfactor score. However since both the factor scores can bedistributed over the 2-dimensional real number space, wefirst converted these scores into a positive number byaddition of a constant of 2.5. We then took the ratio of thepositive factor scores as an indicator of the relative factorscores (factor 1/factor 2).


Characterization of novel exon1-containing transcripts encoded byCCL3Lb (CCL3L2) gene

By using a variety of bioinformatics and molecularapproaches, we generated enough data to support the factthat the described alternative spliced isoforms in Fig. 1c areexpressed and contain the notated sequences as depicted inthe Fig. 1c. Details about the sequences, methods andanalysis can be obtained from [email protected].

References

1. Gonzalez E, Kulkarni H, Bolivar H, Mangano A, Sanchez R,Catano G, et al. The influence of CCL3L1 gene-containingsegmental duplications on HIV-1/AIDS susceptibility. Science2005; 307:1434–1440.

2. Townson JR, Barcellos LF, Nibbs RJ. Gene copy number regulatesthe production of the human chemokine CCL3-L1. Eur J Immunol2002; 32:3016–3026.


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combinatorial content of ccl3l ccl4l copy numbers ...copy numbers inﬂuence hiv-aids susceptibility...

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