Genotype and Allele Frequency of a 32-Base Pair Deletion Mutation In

Original Report

Genotype and Allele Frequency of a 32-Base Pair Deletion Mutation in the CCR5 Gene in Various Ethnic Groups: Absence of Mutation among Asians and Pacific Islanders Yuanan Lu, PhD; * Vivek R. Nerurkar, PhD;* Wan-Mohaiza Dashwood, BS;” Cora L. Woodward, BS;” Sherimay Ablan, BA;” Cecilia M. Shikuma, MD;* Andrew Grandinetti, PhD;” Healani Chang, MPH;* Hien Tran Nguyen, MD;? Zunyou Wu, MD;* Yasuhiro Yamamura, PhD;§ William 0. Boto, PhD;n Andrew Merriwether, PhD;” Takeshi Kurata, MD, PhD;** Roger Detels, MD;++ and Richard Yanagihara, MD*

ABSTRACT nucleotide primer pair designed to discriminate CCR5 alleles without restriction endonuclease analysis. Background: A 32-base pair (bp) deletion mutation in the beta- chemokine receptor CCR.5 gene has been associated with Results: The comparative frequency of CCRUA32 hetero- resistance against human immunodeficiency virus type 1 (HIV- zygosity was 61 of 363 (16.8%) in Caucasians, 17 of 303 (5.6%) 1) infection and disease. Large-scale studies conducted among in Puerto Rican Hispanics, 9 of 490 (1.8%) in Pacific Islanders, Caucasians indicate that individuals who are homozygous for 0 of 606 (0%) in Asians, and 0 of 150 (0%) in Africans. this deletion mutation (A32/A32) are protected against HIV-I infection despite multiple high-risk exposures, whereas CCRS/ Conclusions: The data confirm the high frequency of CCR5/A32 A32 heterozygotes have a slower progression to acquired heterozygosity among Caucasians, Intermediate and low-level immunodeficiency syndrome (AIDS). A32 allele frequencies among Puerto Rican Hispanics and Hawaiians could be attributed to recent European Caucasian Objective: To determine the genotype and allele frequencies of gene flow. By contrast, the inability to detect the A32 allele the CCR5 gene 32-bp deletion mutation among ethnically among Asians and other Pacific Islander groups suggests that diverse non-Caucasian populations. other mechanisms are responsible for resistance to HIV-l infection in these populations. Methods: DNA, extracted from blood collected between 1980 and 1997 from 1912 individuals belonging to various ethnic Key Words: chemokine recepfol; human immunodeficiency groups, including 363 Caucasians, 303 Puerto Rican Hispan- virus, Melanesia, Micronesia, polymerase chain reaction, ics, 150 Africans, 606 Asians, and 490 Pacific Islanders, were Polynesia, population genetics analyzed for the CCR.5 gene 32-bp deletion mutation by a polymerase chain reaction (PCR)-based assay, using an oligo- Int J Infect Dis 1999; 3:186-191.

Presented in part at the 16th Annual Meeting of the American Society “Retrovlrology Research Laboratory, Hawaii AIDS Research Consortium, for Virology, Bozeman, Montana, July 19-23,1997; Fourth International and Native Hawaiian Health Research Program, Pacific Biomedical Conference on AIDS in Asia and the Pacific, Manila, Philippines, Octo- Research Center, University of Hawaii at Manoa, Honolulu, Hawaii; ber 25-29,1997; Fourth Asia-Pacific Congress of Medical Virology Seoul, +Department of Hygiene, Environment and Epidemiology, Hanoi Med- Korea, November 3-5, 1997; and Sixth RCMI International AIDS Sym- ical College, Hanoi, Vietnam; +National Center for AIDS Prevention and posium, San Juan, Puerto Rico, November 15-17,199s. Control, Chinese Academy of Preventive Medicine, Beijing, People’s Supported by U.S. Public Health Service grants G12RR/AI-03061, Republic of China; “AIDS Research Program, Ponce School of Medicine, G12RR/AI03050, and P2ORR/AI-11091 from the Research Centers in Ponce, Puerto Rico; “Center for International Biomedical Research, City Minority Institutions Program, National Institutes of Health, and fund- University of New York, New York, New York; Department of Anthro- ing from the Japanese Foundation for AIDS Prevention and the World pology, University of Michigan, Ann Arbor, Michigan; **Department of AIDS Foundation Sherimay Ablan was supported in part by a grant Pathology, National Institute of Infectious Diseases, Tokyo, Japan; and (R25GM56930) to the Haumana Biomedical Program at the University ++Department of Epidemiology, School of Public Health, University of of Hawaii at Manoa. California at Los Angeles, Los Angeles, California. Received: January 7,1999; Accepted: April 2,1999. This study, which was approved by the Committee on Human Subjects of the University of Hawaii at Manoa, is a collaborative project of the Address correspondence to Dr. Yuanan Lu, Retrovirology Research RCMI Gateway Consortium, comprised of the University of Hawaii at Laboratory, Leahi Hospital, 3675 Kilauea Avenue, Honolulu, Hawaii Manoa, Ponce School of Medicine, and City University of New York. 96816. E-mail: yluQpbrc.hawaii.edu.

186 Population Genetics of CCRS Polymorphism / Lu et al 187

Entry of human immunodeficiency virus type 1 (HIV-l) Genomic DNA Extraction into target cells requires the binding of the external enve- Extraction of genomic DNA from peripheral blood lope glycoprotein gp120 to both the CD4 molecule and mononuclear cells (PBMC), huffy coat samples, or whole one of several chemokine receptors, recently discovered blood blotted onto filter paper was performed in a to function as coreceptors.l-’ T-cell line-tropic HIV-l biosafety laminar-flow cabinet. For the former, PBMC pel- strains utilize the a-chemokine receptor CXCR4,’ whereas lets were resuspended in 50 to 250 yL of cell lysis buffer the B-chemokine receptor 5 (CCR5), which is expressed containing 1 X PCR buffer, 2.5 mM MgCl,, 0.5% NP-40, on monocytes/macrophages, ‘I’ cells, and granulocyte pre- 0.5% Tween-20, and 120 pg/mL proteinase K. Mixtures cursors, is the key cofactor for macrophage-tropic HIV-l were then incubated at 56”~ for 60 minutes, heated at strainszm6which predominate during the asymptomatic 100°C for 10 minutes, and stored at 4°C for later use. phase of infection. DNA extraction from buffy coat samples was accom- A 32-base pair (bp) deletion mutation (A32) within plished using a commercially available kit (Promega, Madi- the second extracellular loop-encoding region of the son, WI), and extraction from filter paper-blotted blood CCRS gene, which results in a truncated, nonfunctional samples, collected from HIV-l-infected individuals in the protein, has been associated with relative resistance to People’s Republic of China and Vietnam, was performed HIV-l infection and slower progression to acquired according to a previously reported technique.16 Briefly, fil- immunodeficiency syndrome (AIDS).‘-” Specifically, ter paper-blotted blood samples were minced with sterile A32/A32 homozygotes are protected against acquisition scissors and transferred to a 1.5 mL Eppendo@centrifuge of HIV-l by the mucosal route despite high-risk expo- tube (Brinkmann Instruments Inc., Wesbury, NY) con- sure, whereas disease progression among CCR5/A32 taining 300 p,L of 100 mM NaCl, 10 mM Tris-HCl (pH heterozygotes occurs more slowly. 8.0) 75 kg proteinase K, 0.5% sodium dodecyl sulfate, and Among Caucasians, the genotype frequencies of 1 mM ethylenediaminetetraacetic acid. Following incuba- CCR5/A32 heterozygosity and A32/A32 homozygosity tion with occasional gentle inversion at 56’C for 1.5 hour, are 16 to 35% and 1.0 to 3.6%, respectively.7-9z11Limited genomic DNA was isolated using the phenol-chloroform information is available on the A32 allele frequency method with ethanol precipitation. among non-Caucasian populations.7,9~12-15To address this issue, the authors developed a polymerase chain reaction CCR5 Genotyping by PCR (FCR)-based assay to determine the genotype and allele frequencies of this CCR5 polymorphism in nearly 2000 A targeted region of the CCR5 gene flanking the 32-bp blood samples collected from various populations, par- deletion was amplified by PCR, using an oligonucleotide ticularly those in Asia and the circum-Pacific region. primer pair designed to discriminate between CCR5 alleles without restriction endonuclease digestion: forward, 5’-GTCTCTCCCAGGAATCATCTTTACCAGATCTC-3’; MATERIALS AND METHODS reverse, 5’-‘ITAGGATICCCGAGTAGCAGATGACCATGACA- 3’. Each PCR was conducted in a final volume of 50 FL Study Population containing 0.2 to 0.5 i.i,g template DNA, 1 X PCR buffer, Blood samples, collected from 1980 to 1997, from 1912 200 PM each dN= 1 mM MgCl,, 1.25 U Themus aquati- individuals (1077 HIV-l-infected and 835 uninfected) cus DNA polymerase (Perkin-Elmer Corporation, Norwalk, belonging to various ethnic groups were studied: 187 CT), and 50 nM each primer. Thermocycling conditions HIV-1seropositive (48 male and 139 female) and 176 HIV- consisted of 45 cycles with an initial denaturation at 94’C 1-seronegative (2 male and 174 female) Caucasians from for 5 minutes, followed by denaturation at 94°C for 1 Hawaii; 303 HIV-l-infected Puerto Rican Hispanics; 150 minute, annealing at 60”~ for 1 minute, and extension at Africans from Uganda; 606 Asians (279 Vietnamese, 128 72°C for 1 minute. The mutated and unmutated alleles Chinese, 109 Japanese, 42 Indian, 39 Filipino, 9 Korean); appeared as 147-bp and 179-bp amplicons, respectively, and 490 Pacific Islanders (253 Polynesians/part-Polyne- which were easily distinguishable by ethidium bromide- sians from Hawaii, 216 Melanesians from New Britain, stained gel electrophoresis using 3% NuSieve agarose and 21 Micronesians from Guam) with or without HIV-l (FMC BioProducts, Rockland, ME). infection. Samples were collected in conjunction with To minimize sample carryover, all specimens and participation in clinical trials or other studies, as in the reagents were handled in a BSL-2 cabinet or UV-mounted HIV-l sentinel surveillance program in Vietnam and the PCR Workstation (C.B.S. Scientific Co., Del Mar, CA). longitudinal registry for cervical and ovarian cancer Reagents were dispensed using aerosol-resistant pipette among women in Hawaii. Demographic data, retrieved tips. Pre-PCR,thermocycling and post-PCR manipulations by coded identifiers, were provided by the respective were performed in physically separated cubicles. Addi- study coordinators. tionally, two negative controls were used in each PCR run. 188 International Journal of Infectious Diseases / Volume 3, Number 4, 1999

Validation of CCR5 Genotyping Assay Beverly, MA), incubated for at least 2 hours at 37°C for PstI and at 50°C for ApoI. Since the single recognition To verify that the 147-bp amplified segment was trun- site of ApoI was within the 32-bp deletion, the presence cated because of the 32-bp deletion, randomly selected of the deletion mutation resulted in ApoI resistance and PCR amplicons were analyzed by restriction endo- P&I sensitivity. nuclease digestion, using two enzymes, ApoI and P&I, To further confirm the specifkity of the PCR-based which made single cuts within and upstream of the 32- CCRS assay, representative PCR products were cloned bp deletion, respectively (Figure 1, A and B). Each diges- using the TA cloning kit (Invitrogen, San Diego, CA), and tion was performed in a 3O+L volume containing the clones were sequenced in both directions on an auto- appropriate digestion buffer, 10 PL PCR amplicon, and mated sequencer (model 373A, Applied Biosystems Inc., 5 units of either ApoI or PstI (New England Biolabs, Inc., Foster City, CA) using the same oligonucleotides

Figure 1. PCR amplification and restriction endonuclease analysis of CCR5 gene. A, Schematic representation (top) and electrophoretic analysis (bottom) of cleavage products of the CCRS gene by Apol (cleavage site indicated by closed arrow). Amplified fragments (CCRUCCRS: lanes 1 and 5; CCRUA32: lanes 2 and 6; A32/A32: lanes 3 and 7) were size fractionated on 3% NuSieve agarose gel before (lanes l-3) and after (lanes 5-7) Apol digestion. Lanes 4 and 8 are 50.bp DNA ladders. 6, Schematic representation (top) and agarose gel (bottom) showing the cleavage products of CCR5 gene before and after fstl digestion. Lanes 1 and 2 represent uncut CCRSKCR5 and CCR5/A32, and lanes 4 to 6 are digested CCRUA32, CCRS/CCR5, and A32lA32. Lane 3 is the 50-bp DNA ladder. C, 3% NuSieve agarose gel showing wild-type CCRUCCRS genotype (lanes 1-l 5) amplified from HIV-1 -negative Melanesians from New Britain. Lanes 16 and 17 are the CCR5/A32 and CCRWCCR5 controls, respectively. D, PCR amplification of the CCR5/A32 heterozygous genotype in Puerto Rican Hispanics (lanes 1-5) and Caucasians (lanes 9-15). Lanes 6 and 16 are the 50.bp DNA ladder, and lane 7 is the CCR5/CCR5 control. Population Genetics of CCR5 Polymorphism / Lu et al 189

employed in PCR. Four randomly selected samples from of these 13 individuals had relatively high CD4 counts each ethnic group were sequenced. Computer-assisted (>500 cells/mm3) and low plasma viral loads (genotypes without restriction enzyme Melanesian descent with no known European Caucasian analysis. Two PCR products, 179 bp and 147 bp in length, admixture, the A32 allele was not found (see Table 1). A representing the CCR5 wild-type and A32 mutated alle- A32 allele frequency of 1.8% (9/506) and a CCR5/A32 les, respectively, were easily discriminated by agarose gel genotype frequency of 3.6% (9/253) was found among electrophoresis (see Figure 1). Analyses of the two frag- Hawaiians, but all nine CCR5/A32 heterozygous Hawai- ments by restriction endonuclease digestion and by ians had Caucasian parents or grandparents. cloning and DNA sequencing verified the 32-bp deletion within the shorter amplified product. Specifically, the 147- bp product was resistant to ApoI digestion, but sensitive DISCUSSION to PstI (see Figure 1 ,A and B), and DNA sequencing indicated that bases 580 to 611 of the CCR5 coding region The discovery of a 32-bp deletion mutation in the CCR5 were missing in the 147-bp amplicon (d&a not shown). gene marks a significant step toward understanding the Among Caucasians, the overall frequency of the het- critical relation between chemokine receptors and HIV-l erozygous genotype (CCR5/A32) was 16.8% (61/363), entry and subsequent disease progression. Genotypic with a A32 allele frequency of 9.2% (67/726). No differ- analysis of this 32-bp deletion mutation among individu- ence was found in the CCR5/A32 genotype frequency als of European Caucasian descent indicates that approx- between HIV-l-infected (32/187 [ 17.1%]) and HIV-l-un- imately 1 to 3% carry two defective alleles and 15 to 30% infected (29/176 [16.5%]) Caucasians (Table 1). By con- are heterozygous. 7-9,11Since polymorphisms of chemokine trast, the frequency of the homozygous genotype receptor genes appear to result in differential resistance (A32/A32) was 1.7% (3/176) among HIV-l-uninfected and susceptibility to HIV-l disease progression, under- Caucasians and nil (O/187) among HIV-l-infected Cau- standing the allele frequencies of the CCR5 gene 32-bp casians (see Table 1). deletion mutation in various ethnic populations may pro- Four Caucasians who possessed the heterozygous vide insights into the evolution of the defective allele, genotype (CCR5/A32) were long-term survivors (infected and in designing effective chemotherapeutic strategies for 11 to 13 y), and 13 other individuals (10 Caucasians, directed against modulating the HIV-l-CCR5 interaction. 1 Hispanic, 1 Japanese, and 1 Pacific Islander) infected Using a rapid PCR-based assay,nearly 2000 DNA sam- with HIV-l for 13 to 17 years were neither heterozygous ples from Caucasians, Puerto Rican Hispanics, Africans, nor homozygous for the 32-bp deletion mutation. Some Asians, and Pacific Islanders were examined for the A32

Table 1. Genotype Frequency of CCR5 Gene 32-bp Deletion Mutation in Various Ethnic Groups HIV- 1-Infected HIV- 1-Uninfected Ethnic Group Total CCR5KCR5 CCR5/A32 A32/A32 Total CCRS/CCR5 CCR5/A32 A32/A32 Asian 408 408 (1.000) 0 (0.000) 0 (0.000) 198 198 (1.000) 0 (0.000) 0 (0.000) Chinese 103 103 (1.000) 0 (0.000) 0 (0.000) 25 25 (1.000) 0 (0.000) 0 (0.000) Japanese 20 20 (1.000) 0 (0.000) 0 (0.000) 89 89 (1.000) 0 (0.000) 0 (0.000) Vietnamese 279 279 (1.000) 0 (0.000) 0 (0.000) 0 Filipino 6 6 (1.000) 0 (0.000) 0 (0.000) 33 33 (1.000) 0 (0.000) 0 (0.000) Indian 0 42 42 (1.000) 0 (0.000) 0 (0.000) Korean 0 9 9 (1.000) 0 (0.000) 0 (0.000) Puerto Rican 303 286 (0.944) 17 (0.056) 0 (0.000) 0 Caucasian 187 155 (0.829) 32 (0.171) 0 (0.000) 176 144 (0.818) 29 (0.165) 3 (0.017) African 150 150 (1.000) 0 (0.000) 0 (0.000) 0 Pacific Islander 29 28 (0.966) 1 (0.034) 0 (0.000) 461 453 (0.983) 8 (0.017) 0 (0.000) Polynesian 28 27 (0.964) 1 (0.036) 0 (0.000) 225 217 (0.964) 8 (0.036) 0 (0.000) Melanesian 1 1 (1.000) 0 (0.000) 0 (0.000) 215 215 (1.000) 0 (0.000) 0 (0.000) Micronesian 0 21 21 (1.000) 0 (0.000) 0 (0.000) Frequencies are shown in parentheses, 190 International Journal of Infectious Diseases / Volume 3, Number 4, 1999

allele. The approximately 17% CCR5/A32 heterozygosity Melanesian populations, does not preclude, in such eth- rate detected among HIV-l-infected and uninfected Cau- nic groups, the existence of other polymorphisms of casians and the A32/A32 homozygosity rate of 1.7% in chemokine receptor genes associated with slower pro- HIV-1-seronegative Caucasians in this study fell within gression to AIDS. For example, the extraordinarily high fre- the range reported previously for Caucasian popula- quencies of the SDP13’A allele among non-Austronesian tions.7-9z11Despite the apparent resistance afforded by highlanders of Papua New Guinea and aboriginal popu- the CCR5 gene 32-bp deletion mutation, protection lations of Australia warrants careful clinical correlation.zs against HIV-1 infection is not absolute, as evidenced by reports of HIV-l infection among A32/A32 homozy- gates.“-‘” Thus far, however, acquisition of HIV-l infection ACKNOWLEDGMENTS in such individuals has been by the parenteral, rather than mucosal route, suggesting utilization of the CXCR4 The authors thank Ms. Caroline Li, Mr. Chris Trujillo, and Mr. coreceptor by dual-tropic strains of HIV-1 among infected Jason Tongson for technical assistance. A32/A32 homozygotes. 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