Genetics and Molecular Biology, 42, 3, 574-577 (2019)
Copyright © 2019, Sociedade Brasileira de Genética.
DOI: http://dx.doi.org/10.1590/1678-4685-GMB-2018-0243
Short Communication
Perforin gene PRF1 c.900C> T polymorphism and HIV-1 vertical transmission
Luisa Zupin1 , Vania Polesello2, Anselmo Jiro Kamada3, Rossella Gratton2, Ludovica Segat2, Louise
Kuhn4 and Sergio Crovella1,2
1
Department of Medicine, Surgery and Health Sciences, University of Trieste, Trieste, Italy.
Institute for Maternal and Child Health IRCCS “Burlo Garofolo”, Trieste, Italy.
3
Departmento de Genética, Universide Federal de Pernambuco, Recife, PE, Brazil.
4
Gertrude H. Sergievsky Center and Department of Epidemiology, Mailman School of Public Health,
Columbia University, New York, NY, USA.
2
Abstract
Perforin-1, a component of the immune system, is able to control Human Immunodeficiency Virus-1 (HIV-1) replication and could be involved in HIV-1 mother-to-child transmission (MTCT). This study aims at evaluating the role of the
c.900C > T PRF1 gene (encoding for perforin-1) polymorphism (rs885822) in HIV-1 MTCT. The PRF1 c.900C > T
polymorphism was genotyped in 331 children from Zambia using a Taqman probe on a Real Time PCR platform. The
PRF1 c.900C > T C/T genotype was more frequent among HIV-1 exposed but non-infected children than in HIV-1
positive cases, and the results were confirmed among children infected during breastfeeding. PRF1 c.900C > T correlated with protection against HIV-1 MTCT, suggesting its role in HIV-1 vertical transmission.
Keywords: PRF1, perforin, HIV-1 susceptibility.
Received: August 22, 2018; Accepted: February 04, 2019.
Perforin-1 (pore forming protein) is a protein present
in the granules of cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells (Heintel et al., 2002). In the presence
of calcium, Perforin-1 polymerizes and forms channels in
the target cell membrane through which other components
of lytic granules including granzyme A, granzyme B and
granulysin may enter in the cells (Shresta et al., 1998;
Stenger et al., 1999). Therefore, Perforin-1 is one of the
fundamental components of the death machinery of CTLs.
CTLs possess anti-viral activity and in Human Immunodeficiency Virus-1 (HIV-1) infection they could concur in the
control of viremia both during the initial and the persistent
phases of infection (Musey et al., 1997; Ogg et al., 1998).
The fraction of perforin-expressing HIV-1 specific CD8+ T
cells inversely correlates with the peripheral blood CD4+ T
cell count thus being a marker for disease progression
(Shresta et al., 1998).
Perforin-1 expression in ex vivo HIV-specific CD8+
T cells was described as higher in healthy controls compared to patients with uncontrolled viral replication, and an
inverse correlation between perforin-1 expression in HIVspecific CD8+ T cells and viral load was observed (Migueles et al., 2008; Hersperger et al., 2010). Perforin-1 is
encoded by the PRF1 gene, located at 10q22, and one polySend correspondence to Luisa Zupin. Department of Medicine,
Surgery and Health Sciences, University of Trieste, Trieste, 34100,
Italy. E-mail: luisa.zupin@burlo.trieste.it.
morphism, namely c.900C>T (rs885822), was previously
associated with HIV-1 vertical transmission in a Brazilian
population (Padovan et al., 2011). In the present study,
PRF1 c.900C>T was analyzed in a population of HIV-1 exposed (infected and uninfected) children from Zambia in
order to replicate previous findings, contributing to disclose its possible involvement in HIV-1 mother to child
transmission (MTCT).
In this study, a subset of the population enrolled in the
Zambia Exclusive Breastfeeding Study (ZEBS, Lusaka
Zambia ClinicalTrials.gov Identifier: NCT00310726) was
recruited. Briefly, ZEBS is a randomized clinical trial implied in the investigation of the relationship between the
time of breastfeeding (i.e. exclusive breastfeeding up to 4
months, or breastfeeding with a median of 16 months) and
the risk of HIV-1 transmission and child mortality. Between May 2001 and September 2004, 958 HIV-1 positive
women were followed during pregnancy up to the delivery
and until 24 months postpartum (PP) with their infants.
Newborns were tested for HIV-1. All women were counseled to breastfeed to at least 4 months, and then half of
them were randomized to stop breastfeeding and the other
half to continue it. Women received only a single-dose
nevirapine as prophylaxis to prevent HIV-1 MTCT.
For this analysis, 331 infants were selected, 22 had
intrauterine (IU) HIV-1 transmission (defined as a positive
polymerase chain reaction (PCR) result within 2 days of
Zupin et al.
575
birth), 25 had intrapartum (IP) HIV-1 MTCT (defined as a
positive PCR result within 42 days of birth with an earlier
negative result), and 38 had postnatal (breastfeeding)
HIV-1 MTCT (defined as a positive PCR results older than
42 days with a negative earlier result in a breastfed child),
246 were HIV-1 exposed but uninfected children (designed
as HIV-). All women provided a written informed consent
allowing children to participate in the study. All the experiments and procedures have been performed in accordance
with ethical standards of the 1975 Declaration of Helsinki
(7th revision 2013) and the Ethics Committee of IRCCS
Burlo Garofolo approved the research (protocol L-1106 1
May 2010).
DNA was extracted as reported by Segat et al. (2014).
The PRF1 polymorphism was detected using TaqMan
SNPs genotyping C___1799201_10 assay and TaqMan®
GTXpress Master Mix with the ABI7900HT Real Time
PCR platform (Applied Biosystems - Life Technologies,
Carlsbad, CA U.S.A.) following the manufacturer’s instructions.
The PRF1 allele and genotype frequencies were calculated by direct counting. Fisher’s exact test was used for
pairwise comparison of allele and genotypes. Logistic regression and Wald’s test were conducted to examine the association between polymorphism genotypes and the risk of
HIV-1 MTCT. The statistical tests were performed with the
free software R version 3.1.3 (R Core Team, 2018). Posthoc power calculations were performed with G*Power
software version 3.1.9.2 using post-hoc calculation employing Fisher’s exact test (Faul et al., 2007).
The PRF1 c.900C > T C/T genotype was more frequent among HIV- compared to HIV+ children, and was associated with decreased risk of acquiring HIV-1 infection
(p=0.03, OR=0.47, CI=0.23-0.94; power=0.68; Table 1
and Table S1) also after adjustment for maternal CD4 cells
count and HIV-1 plasma viral load (p=0.01, OR=0.40,
CI=0.19-0.81; data not shown). When children were subdivided according to the route of transmission, C allele and
C/T genotype correlated with protection towards HIV-1
MTCT in the group of PP infected children (C allele:
p=0.02, OR=0.35, CI=0.11-0.90; power=0.64; and C/T genotype: p=0.01, OR=0.22, CI=0.04-0.74; power=0.50; Table 1 and Table S1) also after adjustment for maternal CD4
cells count and HIV-1 plasma viral load (p=0.009,
OR=0.19, CI=0.05-0.66; data not shown).
Our results partially agree with those of Padovan et
al. (2011). In fact, both studies observed an increased frequency of c.900C>T T allele in the HIV-1 positive children
group if compared to the group of HIV-1 exposed but not
infected children. Our study found the c.900C>T T/T geno-
Table 1 - PRF1 polymorphism allele genotype frequencies (and counts) in HIV-1 exposed but not infected children (HIV-) and HIV-1 infected children
(HIV+) stratifying for timing of HIV-1 mother to child transmission (MTCT) in intrauterine (IU) intrapartum (IP) and postpartum (PP) groups.
Children
HIV+
IU
IP
PP
HIV-
HIV+ vs.
HIV-
IU vs. HIV-
IP vs. HIV-
PP vs. HIV-
n=85
n=22
n=25
n=38
n=246
T
0.89 (151)
0.82 (36)
0.88 (44)
0.93 (71)
0.83 (410)
C
0.11 (19)
0.18 (8)
0.12 (6)
0.07 (5)
0.17 (82)
Ref.
Ref.
Ref.
Ref.
T/T
0.81 (69)
0.73 (16)
0.76 (19)
0.87 (34)
0.70 (171)
C/T
0.15 (13)
0.18 (4)
0.24 (6)
0.13 (3)
0.28 (68)
p=0.03;
p=0.60;
p=0.82;
p=0.01;
CI=0.23-0.94; CI=0.15-2.05; CI=0.25-2.18; CI=0.04-0.74;
OR=0.47
OR=0.63
OR=0.79
OR=0.22
C/C
0.04 (3)
0.09 (2)
0.00 (0)
0.004 (1)
0.03 (7)
p=1.00;
p=0.19;
CI=0.17-4.82; CI=0.28-17.83
OR=1.06
; OR=3.03
HWE
c2=4.48
p=0.03
c2=3.33
p=0.07
c2=0.46
p=0.49
c2=4.86
p=0.03
c2=0.01
p=0.94
PRF1
c.900C > T
rs885822
HWE = Hardy Weinberg equilibrium
HIV- = HIV-1 exposed but not infected children
HIV+ = HIV-1 infected children
IU = intrauterine HIV-1 mother to child transmission
IP = intrapartum HIV-1 mother to child transmission
PP = postpartum HIV-1 mother to child transmission
OR = odds ratio
CI = confidence interval
n.c. = not calculable
p=0.11;
p=0.83;
p=0.54;
p=0.02;
CI=0.35-1.09; CI=0.43-2.55; CI=0.23-1.68; CI=0.11-0.90;
OR=0.63
OR=1.11
OR=0.68
OR=0.35
Ref.
Ref.
Ref.
n.c.
Ref.
p=1.00;
CI=0.01-5.90;
OR=0.72
576
type to be more frequent among HIV+ respect to T/C genotype, while in the study of Padovan et al. (2011) T/T was
more frequent compared to C/C homozygous genotype.
The study of McIllroy et al. (2006) also analyzed this PRF1
gene polymorphism in a cohort of French HIV+ seroconverters. They observed that PRF1 c.900C>T polymorphism seemed not to alter the amino acidic sequence of
perforin-1 protein and was not associated with HIV-1 infection or progression. The different mode of HIV-1 transmission and different ethnic genomic background could
account for the divergent results.
In the current study, we observed an association of
PRF1 polymorphism with the susceptibility to HIV-1 in the
HIV+ group, but intriguingly, it was confirmed only in the
infants that presented PP MTCT, thus indicating a protective effect of the variants at birth and not during the pregnancy or the delivery.
The functional effect of this polymorphism on the
protein and its possible influence in HIV-1 vertical transmission were not yet reported. A hypothesis suggested by
Padovan et al. (2011) indicated the PRF1 c.900C>T polymorphism as exerting possible effects on protein expression, which might in turn influence NK functionality. Indeed, the NK response plays a pivotal role in preventing
HIV-1 vertical transmission, as a higher HIV-1 specific NK
response was found in HIV-1-infected non transmitter
mothers and exposed-uninfected children compared to
transmitter mothers and exposed-infected children
(Tiemessen et al., 2009). However, this speculation should
be confirmed by functional analysis, which have not been
performed in our study due to the fact that the sole biological material available were dried blood spots.
We are aware that the small sample size of our population could have influenced the statistical analysis, especially in the subgroups classified according to the route of
MTCT. However, the quite high power of the statistically
significant associations allows us to be confident about the
statistical relevance of the results. We also decided to not
perform corrections for multiple testing in order to unravel
all the possible associations that could be significant, especially in an infectious disease where role of genetic
polymorphisms should be small, and since after applying
multiple test corrections our significance will be lost.
Another point that should be taken into account is the
MTCT, as even when the viral HIV-1 RNA is undetectable,
the risk MTCT still exists (see for example Reliquet et al.,
2014). However, the modern test for virologic diagnosis
did not reveal possible infections, so possibly creating a
bias in our analysis.
Considering our findings and the comparison with the
two other studies analyzing the role of PRF1 variants in the
context of HIV-1 infection, further association studies in
populations of different ethnic backgrounds are necessary
to disclose the effective role of perforin-1 in HIV-1 MTCT
susceptibility.
PRF1 and HIV-1 MTCT
Acknowledgments
This work has been financially supported by RC08/17
grant from IRCCS Burlo Garofolo Trieste / Ministry of
Health (Italy). This study was also supported in part by
grants from the Eunice Kennedy Shriver, National Institute
of Child Health and Human Development (NICHD), National Institutes of Health (NIH) (HD39611, HD40777,
HD57617). VP was the recipient of a fellowship from
IRCCS Burlo Garofolo.
Conflict of interest
The authors declared no conflict of interest.
Author contributions
LZ conducted the experiments and wrote the manuscript; VP participated in performing the experiments; AJK
conducted the statistical analyses and participated in writing the manuscript; RG participated in performing the experiments; LS critically revised the manuscript and supervised the experiments; LK was responsible of the
management of the patients and collected the specimens;
SC conceived the study and critically revised the manuscript. All authors read and approved the final version.
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Supplementary material
The following online material is available for this article:
Table S1 - The results from power analysis (Fisher’s exact test).
Associate Editor: Mara H. Hutz
License information: This is an open-access article distributed under the terms of the
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distribution and reproduction in any medium, provided the original article is properly cited.