Egyptian Pediatric Association Gazette 66 (2018) 121–124

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Egyptian Pediatric Association Gazette

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Angiotensin-converting enzyme polymorphism and pediatric pneumonia T

a,⁎ b a a
Nihal El Rifai , Hanan Alwakeel , Hala Shaaban , Mohamed Abdelfattah
a
Department of Pediatrics, Faculty of Medicine, Cairo University, Egypt
b
Department of Clinical and Chemical Pathology, Faculty of Medicine, Cairo University, Egypt

A R T I C LE I N FO A B S T R A C T

Keywords: Objective: Pneumonia is one of the leading causes of death for the Pediatric age group under five years
Angiotensin-converting enzyme worldwide. The role of the angiotensin-converting enzyme (ACE) insertion/deletion (I/D) polymorphism in the
Pediatric risk and outcome of community-acquired pneumonia (CAP) has been studied in different ethnic populations and
Pneumonia yielded inconsistent results. Therefore, the present study aimed to investigate whether this polymorphism is
Polymorphism
associated with the risk and outcome of CAP in the Egyptian pediatric population.
Methods: The prospective observational case-control study was performed on 77 children aged 2 months to 12yrs
hospitalized with CAP and 73 matched healthy controls. Candidates were subjected to clinical and radiological
evaluation in addition to complete blood count, c-reactive protein and genotyping for the ACE I/D poly-
morphism using PCR technique. Follow up of the outcome in the form of need for oxygen, ICU admission, need
for mechanical ventilation, duration of hospital stay and death was done for all patients.
Results: No statistically significant differences was observed between pneumonia patients and controls regarding
the different genotypes of the ACE polymorphism II, ID and DD genotypes (p = 0.773). No association was
detected between the different genotypes of the ACE polymorphism II, ID and DD genotypes and the need for
oxygen, ICU admission, need for mechanical ventilation, duration of hospital stay and death (P = 0.143, 0.527,
0.716, 0.288, 0.544).
Conclusion: The ACE I/D polymorphism is not associated with the risk nor the outcome of pneumonia in our
pediatric population.

Introduction (17q23). A polymorphism involving the presence (insertion, I) or ab-

Community acquired pneumonia (CAP) and its critical complica-
tions have been highlighted in previous studies that succeeded in the
discovery of polymorphic variants of some host genes associated with
the diversity in the response to CAP.1 The ACE gene I/D polymorphism
and its association with the pathogenesis and outcome of pneumonia
has been studied in different ethnic populations and the results were
contradictory.2–5 ACE is a zinc metallopeptidase widely distributed on
the surface of endothelial and epithelial cells. ACE converts the inactive
angiotensin I to the active and potent vasoconstrictor angiotensin II
which is the main active product of the renin–angiotensin system.6 Fox
and his colleagues7 reported that ACE inhibitors increase bradykinin
and substance P which together sensitize the sensory nerves of the
airways and enhance the cough reflex. Mortensen et al.8 concluded that
treatment with ACE inhibitors and angiotensin receptor blockers were
both associated with a decreased risk of pneumonia related mortality.
The gene encoding ACE is located on the long arm of chromosome 17
sence (deletion, D) of a 287-bp sequence of DNA is located in intron 16
of the gene.2 Mean ACE activity levels in DD genotype individuals were
approximately twice that found in II genotype. Takahashi et al.9 ob-
served genetic differences in ACE polymorphisms between Asian and
non-Asian patients and stated that polymorphisms I/I and I/D, which
are more prevalent in Asian population, showed a protective trend,
whereas the D/D polymorphism was less protective. The potential loss
of protective effect may be explained by increased levels of serum ACE
and catabolism of kinins in patients with the D/D polymorphism.10In
addition, it was pointed out that the ACE D allele was associated with
decreased ACE inhibitor-related cough and thus, it is biologically
plausible that ACE DD genotype might increase pneumonia ris-
k.11Unfortunately, studies for the Egyptian population especially the
pediatric age are lacking.5 Therefore, we aimed at the current study to
investigate the effect of the ACE polymorphism on the risk and outcome
of CAP in our pediatric patients.

Peer review under responsibility of Egyptian Pediatric Association Gazette.

⁎
Corresponding at: Faculty of Medicine, Cairo University, New University Children’s Hospital (Abu El Reish), 4 – Gamal Salem St. Doki, Cairo, Egypt.
E-mail addresses: hassangaafar@kasralainy.edu.eg (N. El Rifai), hanan.alwakeel@kasralainy.edu.eg (H. Alwakeel).

https://doi.org/10.1016/j.epag.2018.08.001
Received 1 August 2018; Accepted 28 August 2018
Available online 07 September 2018
1110-6638/ © 2018 The Egyptian Pediatric Association Gazette. Publishing services provided by Elsevier B.V. All rights reserved. This is an open access article
under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).
N. El Rifai et al.
Materials and methods
Study design and subjects
The present prospective observational case-control study was con-
ducted at the Cairo University Children’s Hospital, Faculty of Medicine
on 77 patients admitted to the general wards or ICU with the diagnosis
of CAP. Patients’ age ranged from 2 months to 12 years. Diagnosis of
CAP was based on the presence of consolidation or infiltrates on chest
X-ray in addition to at least two of the following criteria: acute cough,
sputum production, tachypnea, chest pain, fever > 38°Celceus
or < 35°Celceus, auscultatory findings consistent with pneumonia,
leukocytosis or leucopenia (> 10 × 109 /L, < 4 × 109 /L, or > 10%
rods in leukocyte differentiation, and CRP greater than three times the
upper limit of normal. Seventy-three age and sex matched healthy
controls were recruited from the follow-up ophthalmology clinic of the
hospital. Exclusion criteria in this study were the presence of malig-
nancy, chronic diseases or respiratory tract infections other than
pneumonia. All patients were subjected to a complete clinical study
(thorough history and physical examination) upon study inclusion, with
emphasis on symptoms and signs of pneumonia; in the form of cough,
sputum production, fever, etc…). Patients were followed up for the
need for oxygen, ICU admission, duration of hospital admission, need
for mechanical ventilation, hospital mortality. Investigations in the
form of: Complete blood counts (CBC) and CRP were performed for
patients. ACE I/D Polymorphism using PCR were performed for all
patients and controls.
Detection of ACE I/D polymorphism using PCR
Genomic DNA of included subjects was isolated from ethylenedia-
mine tetra-acetic acid (GeneJET Whole Blood Genomic DNA
Purification Kit, Thermo Fisher Scientific, Inc.). Genotyping of ACE I/D
Polymorphism was performed using a polymerase chain reaction (PCR).
The primer sequences used were as follows: Forward Primer 5′-CTG
GAG ACC ACT CCC ATC CTT TCT-3′; Reverse Primer: 5′-GAT GTG GCC
ATC ACA TTC GTC AGA T-3′12. Enzymatic amplification was performed
using Dream TaqTM PCR Master Mix (Thermo Fisher Scientific, Inc) in
total volume of 25 μl. For PCR amplification, an initial denaturation at
95 oC for 5 min was followed by 30 cycles consisting of 1 min of de-
naturation at 94 °C, 1 min of annealing at 58 °C and extension for 2 min
at 72 °C and then a final extension at 72 °C for 4 min. Products were
separated on 2% agarose gel and bands were visualized by ethidium
bromide staining under ultraviolet (UV) light. This reaction yielded one
fragment of 490 bp indicating a homozygous genotype for insertion (II),
or one 190 bp fragment indicating a homozygous genotype for deletion
DD while the presence of 490 and 190 bp products indicated hetero-
zygous status (ID) Fig. 1.
Fig. 1. PCR results for ACE I/D polymorphism genotypes. Lane (4): A product of
490 bp indicated a homozygous genotype for insertion (II). Lane (1, 2, 3, 5, 6,
8): A product of 190 bp indicated a homozygous genotype for deletion (DD).
Lane (7, 9, 10): presence of 490 and 190 bp products indicated heterozygous
genotype (D/I). Lane (11): DNA ladder 50 bp.
122
Egyptian Pediatric Association Gazette 66 (2018) 121–124
Ethical considerations
The aim and nature of the study was explained for each parent
before inclusion. An informed written consent was obtained from par-
ents/surrogates before enrollment. The ethical committee of the
Pediatrics department, Faculty of Medicine, Cairo University approved
the work and it conforms to the provisions of the Declaration of
Helsinki in 1964 and its later amendments or comparable ethical
standards.
Sample size
Sample size calculation was performed using Power and Sample Size
Calculator program version 3.0.43. It was based on the following in-
puts: Power of 80%, type I error of 0.05, equal number of candidates in
both cases and controls, true difference in means between groups of
0.12 and standard deviation of 0.26. It was found to be 73 children in
each group (total = 146).
Statistical analysis
Statistical analysis was performed using Minitab version 16 soft-
ware (MINITAB Inc. R, USA). Numerical data were expressed as mean
and standard deviation or median and range as appropriate. Qualitative
data were expressed as frequency and percentage. For quantitative data
that not normally distributed, comparison between two groups was
done using Mann-Whitney test (non-parametric t-test). Chi-square test
was used to examine the relation between qualitative variables. Odds
ratio (OR) with it 95% confidence interval (CI) were used for risk es-
timation. A p-value < 0.05 was considered significant.
Results
Demographic and clinical data of cases
In our study, Patients were 41 male patients (53.2%) & 36 females
(46.8%).They were age and sex matched with 73 healthy children.
75.3% of cases had fever (n = 58) and 74% of cases (57 cases) had
cough. Thirteen cases (16.9%) presented with respiratory distress (RD)
grade I, while 48 cases (62.8%) had RD grade II and 16 cases (20.8%)
presented with RD grade III. Laboratory investigations revealed pre-
sence of shift to the left of neutrophils in 9 patients (11.7% of the cases),
CRP was positive in 26 patients (33.63%) and ESR was elevated in
100% of patients. On radiological assessment, 13 patients (16.9%) had
lobar pneumonia, while 64 patients (83.1%) had bronchopneumonia.
Baseline Characteristics of 77 Patients with CAP in relation to different
genotypes of ACE I/D Polymorphism are represented in Table 1. There
were no statistical significant differences in demographic and clinical
characteristics of the patients with CAP in comparison between dif-
ferent genotypic groups. Fever is more common in the ID and II geno-
types (P = 0.003).
Genotypic results of ACE I/D polymorphism
Genotypic distribution and allele frequencies of ACE I/D poly-
morphism did not differ between cases and control children as shown in
Table 2. Generally, DD represented the highest prevalence followed by
ID then II among cases and control children but this didn’t reach a
statistical significance difference.
Outcome among cases
As regards the outcome among our cases; sixty-two cases (80.5%)
needed oxygen, 12 cases (15.5%) needed ICU admission, 5 cases (6.5%)
were ventilated and only one patient died due to pneumonia. Seventeen
cases (22.1%) needed prolonged hospital admission (more than one
N. El Rifai et al. Egyptian Pediatric Association Gazette 66 (2018) 121–124

Table 1
The Characteristics of Patients with CAP in Relation to Different Genotypes of ACE I/D Polymorphism.
a
Variable DD (N = 37) ID (N = 35) II (N = 5) P value

Sex M/F No (%) 22(59.5)/15(40.5) 15(42.9)/20(57.1) 4(80)1(20) 0.170

Fever No (%) 33(42.9) 20(57.1) 5(10 0) 0.003
b
RD No (%) 0.39
I 6(16.2) 5(14.3) 2(40)
II 21(56.8) 24(68.6) 3(60)
III 10(27) 6(17.1) 0(0)
Cough No (%) 26(70.3) 28(80) 3(60) 0.489
Expectoration No (% 17(45.9) 18(51.4) 3(60) 0.795

Chest X-ray No (%)

Lobar pneumonia 5(13.5) 6(17.1) 2(40) 0.332
Broncho-pneumonia 32(86.5) 29(82.9) 3(60)
c
TLC (×109/L)
Mean (SD) 12.319 ± 5.8 10.59 ± 5.77 14.28 ± 6.21 0.270
d
CRP + ve No (%) 14(37.8) 9(25.7) 3(60) 0.243

a
P value < 0.05 is statistically significant.
b
RD = Respiratory distress.
c
TLC = Total leucocytic count.
d
CRP = C-reactive protein.

Table 2 Discussion
Genotypic Distribution and Allele Frequencies of ACE I/D Polymorphism.
Variable Cases Control P-valuea Odds ratio 95% bCI
Immense number of studies investigated the association between
(N = 77) (N = 73) the ACE I/D polymorphism and numerous diseases. These investiga-
No. (%) No. (%) Lower Upper tions didn’t focus only on the presence or incidence of disease but also
on manifestations, efficacy of therapy, interaction with other genetic or
DD genotype 37 (48.1%) 39 (53.4%) 0.773 0.806 0.495 3.158
ID genotype 35 (45.5%) 21 (28.8%)
environmental factors, recovery rates, disease progression and sur-
II genotype 5 (6.5%) 13 (17.8%) vival.13 The impact of ACE polymorphism on CAP initiation and pro-
D allele 109 99 (67.8%) 0.6 1.14 0.7 1.419 gression possibly through affecting augmentation of the inflammation
(70.8%) process was demonstrated by previous studies.1
I allele 45 (29.2%) 47 (32.2%)
A meta-analysis of 12 studies that systematically evaluated the as-
a
P value < 0.05 is statistically significant. sociation between ACE polymorphism and pneumonia risk pointed to
b
CI = Confidence interval. the fact that the primary articles only provided data for Caucasian and
Asian populations in addition to the presence of only 3 case-control
studies performed for the pediatric age.5To our knowledge, the present
study is one of a very few studies performed in xxx population espe-
cially the pediatric age group to investigate the association between
ACE polymorphism and pneumonia risk and outcome.
The distribution of the DD, ID and II genotypes of the ACE poly-
morphisms showed to be non-significant between cases and controls (P
value = 0.733, odds ratio = 0.81 and 95% CI = 0.495–3.16) with the
DD genotype the most prevalent in the study population. Similarly, the
allelic frequencies showed a similar distribution between patients and
controls as the D allele was the prevalent allele present in 70.78% of
cases and 67.8% of controls and the I allele was present in 29.2% of
cases and 32.2% of controls (P value = 0.6, odds ratio = 1.14 and 95%
CI = 0.7–1.42). In agreement with the current study, a study by Van de
garde et al. in 200 elderly white Dutch population diagnosed with
Fig. 2. clinical severity and disease outcome among different genotypes of ACE pneumonia and their controls revealed a non-significant association
I/D polymorphism in CAP patients (N: 77 cases). between patients and controls with respect to the three genotypes and
the allelic frequencies. However, the ID genotype was the most pre-
week), while the rest of patients were admitted less than one week. valent in this population.4
Disease outcome among different genotypes of ACE I/D polymorphism Furthermore, Sagnella et al.14 stated that the prevalence of the DD
in CAP patients are presented in Fig. 2. No significant difference be- genotype is small in Asian population as compared to white and African
tween the three genotypes and the outcome of the disease was detected population. On the contrary to our results, the meta-analysis by Nie
(P value > 0.005). Even when pooling the ID and II genotypes together et al.5 demonstrated that the DD genotype was a risk factor for devel-
in one group (ID + II) and comparing DD versus (ID + II) and the oping pneumonia in the overall study population where the DD geno-
outcome of pneumonia, it was found that there were no statistically type had 53% increased pneumonia risk as compared with the ID and II
significant differences between the 2 groups (P > 0.05) as regards the carriers. In the subgroup analysis by ethnicity, it was found that the
clinical outcome where 32 (51.6%) and 30 (48.4%) cases needed association between ACE I/D polymorphism and pneumonia risk re-
oxygen, 7 (58.3%) and 5 (41.7%) needed ICU admission, 2 (40%) and 3 mained significant in Asians (OR = 1.87, 95% CI 1.44–2.43,
(60%) were ventilated. Seven (41.2%) and 10 (58.8%) needed pro- p < 0.00001) and Caucasians (OR = 1.34, 95% CI = 1.09–1.65,
longed hospital admission from the DD and ID + II genotypes conse- p = 0.006). Furthermore, in the subgroup analysis based on age, a
quently. Only one patient died from the ID + II genotypes. significant association was observed in the adult population but not in

123
N. El Rifai et al.
the pediatric population.
Regarding outcome of pneumonia in the current study patients, no
association was detected between ACE I/D polymorphism and outcome
of pneumonia. The oxygen requirement, need for ICU and mechanical
ventilation were not different for the three genotypic groups as were for
duration of hospital stay for more than one week and the number of
deaths. Based on the meta-analysis findings by Nie and colleagues5 that
DD genotype was a risk factor for developing pneumonia in the overall
study population and due to the small number of II genotype, we pooled
ID and II genotypes together (ie: ID + II). Even when pooling them
together, no statistical differences were detected. Similarly, the meta-
analysis by Nie et al.5 reported no statistically significant association
between ACE polymorphism and pneumonia mortality risk (OR = 2.68,
95% CI = 0.8–8.9, p = 0.11) explaining that only three studies in-
cluding 376 patients investigated this association and that more studies
are needed to further investigate the effect of the polymorphism on
pneumomia mortality. They highlighted that other factors including
body mass index, complications and medical treatment play crucial
roles in the prognosis of pneumonia. Furthermore, Van de Garde et al. 4
reported the absence of any trend toward an effect of ACE DD genotype
on pneumonia outcome.
ACE polymorphism is not likely a functional variant being located in
a noncoding region of the gene. The polymorphism is in close linkage
disequilibrium to one or more functional polymorphisms determining
phenotypic variant of the enzyme levels. Accordingly, more functional
experiments are required to investigate the effect of this poly-
morphism.15 Furthermore, the complex nature of pneumonia explains
why pneumonia risk may not be dependent in only a single nucleotide
polymorphism in one single gene.
The limited sample size in the present study especially when com-
paring outcome to different genotype groups may explain the null effect
due to lack of power and signifies the need for large scale studies to
verify clearly the comparison.
Finally, the current study serves as a preliminary report that paves
the way for further investigation on a larger scale and with a long-
itudinal design for better assessment of role of ACE I/D polymorphism
in pathogenesis and possibly the outcome of pneumonia in children
taking into consideration functional polymorphisms that are in close
linkage with the ACE polymorphism.
Conflicts of interests
Authors have no conflicts of interests to declare.
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Egyptian Pediatric Association Gazette 66 (2018) 121–124
Funding
This research didn’t receive any specific grant from funding agen-
cies in the public, commercial or not – for profit sectors.
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