Archives of Medical Research 49 (2018) 18e26

ORIGINAL ARTICLE
Influence of SNPs in Genes that Modulate Lung Disease Severity in a Group
of Mexican Patients with Cystic Fibrosis
Emiy Yokoyama,a Margarita Ch
avez-Salda~na,b Lorena Orozco,c Francisco Cuevas,d Jos
e Luis Lezana,e,f
b
Rosa Mar
ıa Vigueras-Villase~ neda,b and Daniel Adrian Landerob
nor, Julio Cesar Rojas-Casta~
a
Departamento de Gen
etica Humana, Instituto Nacional de Pediatr
ıa, Ciudad de M
exico, M
exico
b
Subdirecci
on de Medicina Experimental, Instituto Nacional de Pediatr
ıa, Ciudad de M
exico, M
exico
c
Laboratorio de Inmunogen
omica y Enfermedades Metab
olicas, Instituto Nacional de Medicina Gen
omica, Ciudad de M
exico, M
exico
d
Servicio de Neumolog
ıa, Instituto Nacional de Pediatr
ıa, Ciudad de M
exico, M
exico
e
Laboratorio de Fisiolog
ıa Pulmonar, Hospital Infantil de M
exico Federico G
omez, Ciudad de M
exico, M
exico
f
Asociaci
on Mexicana de Fibrosis Qu
ıstica A.C., Ciudad de M
exico, M
exico
Received for publication February 4, 2016; accepted April 11, 2018 (ARCMED-D-16-00065).

Background. The variation in cystic fibrosis (CF) lung disease not always is explained by
the CFTR genotype, so it has become apparent that modifier genes must play a consider-
able role in the phenotypic heterogeneity of CF, so we investigated the association of
allelic variants in modifier genes that modulate the severity of lung function in a group
of Mexican patients diagnosed with CF.
Methods. We included 140 CF patients classified according to lung phenotype and
analyzed 17 single nucleotide polymorphisms (SNPs) by TaqManÒ allelic discrimination.
Results. We demonstrated that patients with GG or GC genotype of the allelic variant
rs11003125 (MBL2-550) of the MBL2 gene exhibit most of the lung manifestations at
an earlier age; and the rs1042713 allelic variant of ADRB2 gene, showed statistical dif-
ference only with the age of first spirometry. When we used the dominant model, the
MBL2 allele rs11003125 (MBL2-550; p 5 0.022, Odds Ratio (OR) 2.87, 95% CI
1.14e7.27) was significantly associated with CF patients as risk factor, and the ADRB2
allele rs1042713 (p.Arg16Gly; p 5 0.005, Odds Ratio (OR) 0.37, 95% CI 0.19e0.75)
was significantly associated with CF patients as protect factor.
Conclusions. Our findings suggest that the MBL2 and ADRB2 genes exerts an important
genetic influence on the lung disease in our patients. Taking into account our results, we
insist on not leaving aside this type of studies, since having techniques such as GWAS or
WES will be able to advance in achieving a better quality of life for CF patients with se-
vere lung disease. Ó 2018 IMSS. Published by Elsevier Inc.
Key Words: Cystic fibrosis, Gene modifiers, Lung disease, Mexican patients.

Introduction (Asociaci
on Mexicana de Fibrosis Qu
ıstica) approximately

400 children are born with CF each year (2), even a
Cystic fibrosis (CF, OMIM#219700) is the most common
newborn screening conducted in Mexico City reported 2
autosomal recessive lethal disease in Caucasians (1). The
CF-affected newborns among 7139 screened patients
actual incidence in Mexico is still uncertain, however, ac-
(1:3597) (3), and also according to the World Health Orga-
cording to the Cystic Fibrosis Mexican Association
nization (WHO) there are 1:8500 live births CF-affected
(4). CF is caused by mutations in CFTR gene (Cystic
Address reprint requests to: Margarita Ch
avez-Salda~na, Dra., Sub- Fibrosis Transmembrane Conductance Regulator), and
direcci

on de Medicina Experimental, Instituto Nacional de Pediatr
ıa, In-
more than 2,000 different mutations have been detected
surgentes Sur, N

um. 3700-C, Col. Insurgentes-Cuicuilco, 04530, Ciudad
de M
exico, M
exico; Phone: (þ52) (55) 1084-0900, ext. 1453; E-mail: (5). Some of the mutations in this gene produce a partial
mdoloreschs@gmail.com or complete loss of the CFTR protein function, causing a

0188-4409/$ - see front matter. Copyright Ó 2018 IMSS. Published by Elsevier Inc.
https://doi.org/10.1016/j.arcmed.2018.04.010
 Gene Modifiers Associated with Cystic Fibrosis
defect in the transport of electrolytes in the apical mem-
brane of epithelial cells and subsequently leading the pro-
duction of abnormally viscous exocrine secretions (6).
This defect caused CF, which is considered a multisystem,
chronic, progressive and lethal genetic disease, character-
ized by a high clinical variability (7e9).
Differences in lung disease severity are not totally ex-
plained by CFTR genotype since it is the first cause of
morbidity and is responsible for 80% mortality (6). Burke
W, et al. (10), showed not differences among individuals car-
rying two copies of the p.Phe508del mutation (homozygous
patients) grouped in pulmonary function, non-pulmonary
complications or overall clinical severity. These findings sug-
gest that environmental factors or genetic background, known
as heritability, which have been estimated between 0.54 and
1.0 (11), could contribute significantly to the variability of
lung disease. Besides, other factors have been proposed
involved in the physiopathology of CF, like modifier genes
which change the severity of the clinical manifestations
without influencing in the etiology of the disease (12e14).
Several studies have analyzed different modifier genes
focused in lung-disease severity, like those genes involved
in class II major histocompatibility complex, a1-antitrypsin
(a1-AT ), a1-antichymotrypsin (a1-ACT ), lectin, mannose-
binding soluble 2 (MBL2), tumor necrosis factor alpha
(TNF-a), glutathione S-transferase mu-1 (GSTM1), trans-
forming growth factor b-1 (TGFb1), nitric oxide synthase
3 (NOS3), and b-2 adrenergic receptor (ADRB2) (15,16).
In different studies, the results of the association between
variants in different genes and severity of CF lung disease
have been replicated, although in other cases the same as-
sociation has not. Buranawuti K, et al. (17) analyzed geno-
types of TNFa-238, TNFa-308, TGFb1 -509 and MBL2
among patients with cystic fibrosis classified according to
age and survival status; specifically, their results sustain that
MBL2 O/O genotype appear to be a genetic modifier of sur-
vival of cystic fibrosis. Recently, Speletas M, et al. (18)
analyzed the association of MBL2 genotypes with MBL
levels and various morbidities of a neonatal intensive care
unit (NICU), in 134 neonates of NICU (83 term and 51 pre-
term) and 150 healthy neonates; their results support the
notion that neonates with MBL deficiency and low MBL
serum levels at birth may be at higher risk of developing se-
vere respiratory complications. In this context, the aim of
this study was to investigate the association of allelic vari-
ants in modifier genes that modulate the severity of lung
function in a group of Mexican patients diagnosed with CF.
Material and Methods
Study Population
We included 140 of 230 unrelated Mexican patients clini-
cally diagnosed with CF, referred from the Asociaci
on
19
Mexicana de Fibrosis Qu
ıstica (AMFQ), Instituto Nacional
de Pediatr
ıa (INP), and Hospital Infantil de M
exico, geno-
typed previously (19). Ethic and Research Committees
approved this study from all participating institutions. Par-
ents and the patients signed the corresponding informed con-
sent forms. The patients were classified into mild or severe
pulmonary phenotype (PPh) based on: a) the age (years) of
the first isolation of Pseudomonas aeruginosa (P. aerugino-
sa) (median [Md] 5 6.8: severe PPh 5 !6.8; mild PPh 5
O6.9); b) the annual decline in forced expiratory volume
during the first second (FEV1) as determined by spirometry
(measured as the difference between the highest FEV1 and
the lowest FEV1 divided by the number of years) (20)
(Md 5 4.2: severe PPh 5 O4.2; and mild PPh 5 !4.19);
c) isolation of P. aeruginosa at admission (severe
PPh 5 positive for P. aeruginosa; mild PPh 5 negative for
P. aeruginosa); d) assessment using the Brasfield scoring
system (scored as 25 points when chest radiography showed
no damage and as zero points when displayed severe lung
damage) (21) (Md 5 18: severe PPh 5 !18; and mild
PPh 5 O19). We took a sub-group of 101 patients with se-
vere CFTR mutations (both alleles with severe
mutationsemutations Class IeIII) to adjust p-values.
Genotyping
Genomic DNA from 140 patients who had been previously
CFTR genotyped (19), were analyzed for 17 allelic variants
or SNPs from different genes, including a1-AT (S allele:
p.Glu264Val [rs17580], Z allele: p.Glu342Lys [rs28929474],
3’ UTR enhancer [c.G1237A]); a1-ACT (-15Thr-Ala
[rs4934], p.Leu55Pro [rs1800463]); IL10 (c.1082G!A
[rs1800896]); TNFa (c.-308G!A [rs1800629]; c.-238G!A
[rs361525]); MBL2 (MBL2-550 [rs11003125], MBL2-
221G!C [rs7096206], D allele: p.Arg52Cys [rs5030737], B
allele: p.Gly54Asp [rs1800450], C allele: p.Gly57Glu
[rs1800451]); ADRB2 (p.Arg16Gly [rs1042713], p.Gln27Glu
[rs1042714]); NOS3 (p.Asp298Glu [rs1799983]); and GSTP1
(p.Ile105Val [rs1695]). These SNPs were selected because of
their frequency and impact on lung disease at the time that the
project was designed, and that moment, there were no studies
of genome-wide association. The polymorphisms were de-
tected using the TaqManÒ allelic discrimination assay
(Applied Biosystems, Foster City, CA). Polymerase chain re-
action (PCR) was performed using the ABI PRISM 7900 sys-
tem. The primers were added to a PCR mix consisting of
10 ng of genomic DNA, 2.5 mL of TaqMan master mix,
0.125 mL of 20X assay mix, and ddH2O to produce a final
volume of 5 mL. The amplification protocol included dena-
turing at 95 C for 10 min, followed by 40 cycles of denaturing
at 95 C for 15 sec and annealing and extension at 60 C for
1 min. The genotype of each sample was assigned automati-
cally by measuring the allele-specific fluorescence using
SDS 2.2.3 software for allelic discrimination (Applied Bio-
systems, Foster City, CA). The samples were genotyped in
20 Yokoyama et al./ Archives of Medical Research 49 (2018) 18e26

duplicate for all 17 polymorphisms and the genotyping repro-
ducibility was 100%. Genotype distribution for all polymor-
phisms was in Hardy-Weinberg equilibrium (HWE) in cases
and in controls.
The linkage disequilibrium (LD) and common haplotype
patterns of the SNPs of the genes included in the study with
more than one SNP were analyzed in Haploview 4.2 using
SNP genotyping data (22) of our patients with CF; then we
analyzed only patients by pulmonary phenotype (mild or
severe PPh), all this to finally evaluate complex genotypes
(between genes) by the MDR 3.0.2 software (Multifactor
Dimensionality Reduction).
Statistical Analysis
SPSSÒ 16.0 version was used to demonstrate the associa-
tion between the SNPs and lung disease. Fisher’s exact test
or the c2 tests was used for categorical variables, and the
Mann-Whitney U test or the Kruskal-Wallis test was used
for numerical variables. The association between the tested
genotypes and CF was analyzed by correlation/trend and c2
tests under three different models (dominant, recessive, and
additive or codominant). Odds ratios were estimated within
95% confidence intervals. The HardyeWeinberg Equilib-
rium (HWE) p-values were assessed using a chi-square test.
All p-values !0.05 was considered to be significant.
Haplotype association analysis was performed with the
variants located in the same gene. Those variants with a
D’ value equal or greater than 0.8 were considered in link-
age disequilibrium (LD) and haplotypes formed were
compared with in the software Haploview 4.2 (22) of our
patients with CF, and then correlating only patients, be-
tween pulmonary phenotype (mild or severe PPh). We
tested epistasis between variants located in different loci
by a Multifactor Dimensionality Reduction (MDR) method
the MDR 3.0.2 software (23), using the default parameters.
Results
Gender distribution was 50.7% (71/140) female and 49.3%
(69/140) for male. Regarding clinical manifestations, in
53.6% (75/140) of the patients P. aeruginosa was isolated
at admission, 77.1% (108/140) of the patients exhibited
pancreatic insufficiency (PI), and only 7.1% (10/140) of
the patients exhibited meconium ileus (data not shown).
The patients were classified according to their PPh and it
was determined that the age of first P. aeruginosa isolation,
the age of death, and the Brasfield scores were significantly
lower in the group of severe PPh patients than in the group
of mild PPh patients. Also, the annual FEV1 decline, the
average level of chloride in sweat, and the percentage of pa-
tients from which P. aeruginosa was isolated at admission
were higher in those with severe PPh. Finally, PI was more
frequent in severe PPh patients compared with mild PPh pa-
tients (Table 1). Of these 140 patients included, 53 (37.9%)
presented a p.Phe508del homozygous CFTR genotype, and
28 (20%) were p.Phe508del heterozygous (21).
Allelic Association
The frequencies of the alleles analyzed are shown in
Table 2. When we compared these frequencies between
the two PPh groups, we observed a significant association
in two of the 17 alleles, in the rs11003125 variant
(MBL2-550), located in the promoter region of the MBL2

Table 1. Clinical manifestations of 140 CF patients with mild or severe pulmonary disease

Pulmonary disease

Mild (n [ 80) Severe (n [ 60)

Variable Media ± SDa p

Age at onset of clinical manifestations (years) 2.2  3.4 1.2  2.1 0.118
Age at diagnosis (years) 7.3  7.3 5.0  6.1 0.069
Age of first P. aeruginosa isolation (years) 9.9  6.5 5.7  5.9 !0.001
Age of first spirometry (years) 11.1  5.5 10.2  6.3 0.165
Age of death (years) 20.7  6.7 11.7  8.3 !0.001
Brasfield score 18.3  3.0 15.4  2.8 !0.001
Annual decline of FEV1 (%/year) 4.1  3.6 7.4  5.1 !0.001
Sweat chloride 99.3  15.4 104.5  12.6 0.017

Percentageb

Gender (% female) 45.0% (36/80) 58.3% (35/60) 0.128

First isolation (% P. aeruginosa) 33.8% (27/80) 80.0% (48/60) !0.001
Pancreatic Insufficiency (% PI) 67.6% (54/80) 90.0% (54/60) 0.002
Meconium ileus (% positive) 5.0% (4/80) 10.0% (6/60) 0.198
a
Mann-Whitney U Test.
b
Fisher’s Exact Test.
 Gene Modifiers Associated with Cystic Fibrosis 21

Table 2. Frequency of SNPs analyzed according to pulmonary phenotype in Mexican patients

Pulmonar Patients with the Patients with the Patients with the
Gene phenotype Genotype genotype (%)b Genotype genotype (%)b Genotype genotype (%)b p Unadjusteda cp Adjusted

a1AT rs17580
Severe AA 91.7 AT 8.3 TT 0 0.745 0.921
Mild AA 93.8 AT 6.2 TT 0
rs28929474
Severe GG 93.3 GA 6.7 AA 0 0.511 0.503
Mild GG 95.0 GA 3.8 AA 1.2
30 enhancer (c.G1237A)
Severe GG 93.3 GA 6.7 AA 0 0.355 0.479
Mild GG 88.8 GA 11.2 AA 0
a1ACT rs4934
Severe GG 48.3 GA 41.7 AA 10.0 0.662 0.624
Mild GG 43.8 GA 41.2 AA 15.0
rs1800463
Severe TT 100 TC 0 CC 0 - -
Mild TT 100 TC 0 CC 0
IL10 rs1800896
Severe GG 65.0 GA 28.3 AA 6.7 0.318 0.306
Mild GG 52.5 GA 40.0 AA 7.5
TNFa rs1800629
Severe GG 75.0 GA 20.0 AA 5.0 0.354 0.420
Mild GG 83.8 GA 11.2 AA 5.0
rs361525
Severe GG 83.3 GA 13.3 AA 3.4 0.286 0.391
Mild GG 75.0 GA 15.0 AA 10.0
MBL2 rs11003125
Severe GG 25.0 GC 47.5 CC 27.5 0.013 0.043
Mild GG 16.7 GC 71.7 CC 11.6
rs7096206
Severe GG 78.3 GC 21.7 CC 0 0.136 0.185
Mild GG 71.2 GC 22.5 CC 6.3
rs5030737
Severe CC 65.0 CT 35.0 TT 0 0.085 0.048
Mild CC 78.8 CT 21.2 TT 0
rs1800450
Severe AA 35.0 AG 60.0 GG 5.0 0.064 0.644
Mild AA 52.5 AG 40.0 GG 7.5
rs1800451
Severe AA 95.0 AG 5.0 GG 0 0.732 0.710
Mild AA 92.5 AG 7.5 CC 0
ADRB2 rs1042713
Severe GG 55.0 GA 36.7 AA 8.3 0.015 0.060
Mild GG 31.2 GA 51.3 AA 17.5
rs1042714
Severe CC 68.3 CG 26.7 GG 5.0 0.169 0.434
Mild CC 52.5 CG 40.0 GG 7.5
NOS3 rs1799983
Severe GG 8.3 GT 86.7 TT 5.0 0.158 0.195
Mild GG 20.0 GT 75.0 TT 5.0
GSTP1 rs1695
Severe AA 21.7 AG 56.6 GG 21.7 0.539 0.415
Mild AA 30.0 AG 50.0 GG 20.0

c test.
a 2
b
Mild pulmonary phenotype group: 80 patients; severe pulmonary phenotype group: 60 patients.
c
The p-value adjusted was done only with CFTR severe mutations patients.

gene, and the rs1042713 variant (p.Arg16Gly), located in mutations ( p-value adjusted), rs11003125 variant (MBL2-
codon 16 of the ADRB2 gene ( p 5 0.013 and 0.015, respec- 550) had also significant association with PPh, in addition
tively). When we analyzed only patients with CFTR severe to the rs5030737 variant (D allele or p.Arg52Cys) of the
22 Yokoyama et al./ Archives of Medical Research 49 (2018) 18e26

MBL2 gene (Table 2); however, the rs1042713 (p.Arg16-
Gly) of the ADRB2 gene was not statistically significant.
In both analysis ( p-value unadjusted and p-value adjusted),
the other allelic variants displayed no significant differ-
ences, so we will only focus on these three variants with
significant association.
When we analyzed the association between the clinical
manifestations and the allelic variants of MBL2 and ADRB2
genes in the 101 patients with severe CFTR genotype (both
alleles with severe mutations-Class IeIII), we found in the
rs11003125 allelic variant (MBL2-550) significant differ-
ences in age of the first P. aeruginosa isolation
( p 5 0.010), age of first spirometry ( p 5 0.003), and Bras-
field score ( p 5 0.014) (Table 3), demonstrating that pa-
tients with GG or GC genotype, exhibit most of the lung
manifestations at an earlier age and therefore experience
a more severe clinical progression, while the other signifi-
cant allelic variant rs5030737 (D allele or p.Arg52Cys) of
MBL2 gene did not show any statistical significant associa-
tion with the clinical manifestations. The rs1042713 allelic
variant of ADRB2 gene, showed statistical difference only
with the age of first spirometry ( p 5 0.019) (Table 3).
When we used the dominant model, the MBL2 allele
rs11003125 (MBL2-550; p 5 0.022, Odds Ratio (OR)
2.87, 95% CI 1.14e7.27) was significantly associated with
CF patients as risk factor, and the ADRB2 allele rs1042713
(p.Arg16Gly; p 5 0.005, Odds Ratio (OR) 0.37, 95% CI
0.19e0.75) was significantly associated with CF patients
as protect factor; also, when we analyzed only patients with
CFTR severe mutations in the same dominant model, the
MBL2 allele rs5030737 (D allele or p.Arg52Cys;
p 5 0.041, Odds Ratio (OR) 2.56, 95% CI 1.02e6.40),
and the ADRB2 allele rs1042713 (p.Arg16Gly;
p 5 0.018, Odds Ratio (OR) 0.38, 95% CI 0.17e0.86),
were significantly associated with CF patients as risk a pro-
tective factor, respectively. The association value and the
OR of the ADRB2 allele rs1042713 were statistically signif-
icant when assessed using the additive model (GG vs. AA)
but not the recessive model (Table 4). The other genetic
variants tested did not show any association.
When analyzing the linkage desequilibrium (LD) and
common haplotype patterns of all 17 SNPs, for search sys-
tematically novel association, we did not identify haplotype
blocks conformed by variants located in the same gene,
since all D’ values were less than 0.8. The MDR analysis
did not find any statistical gene-gene interaction since al
p values were greater than 0.05 and Cross Validation
(CV) values did not reach 10/10, the grater was 9/10.
Discussion
Cystic fibrosis is a multisystem disease with highly variable
clinical manifestations, which has not been explained only
by the CFTR mutations (24e26), especially the lung disease,
as some patients with severe CFTR mutations are expected to
show also severe lung disease (6,27e31). For decades, the
broad range of lung-disease severity and the susceptibility
to opportunistic infections have been associated with multi-
ple additive effects, such as complex alleles, modifiers genes,
specifically single nucleotide polymorphisms (SNPs) in
genes that are involved in host defense, inflammation, epithe-
lial repair, mucin production and immunological responses
of the airway (7,32), mutations in genes that can mimic
CF phenotypes, and additional effects, such as those influ-
enced by epigenetic and environmental factor (26). Recently,
whole-genome surveys have identified several genes and
chromosomal regions for which a role in CF is not clear at
all (1,33e35). However, in spite of some discordance in
most representative studies, a few genes have been well-
proven to be CF modifiers such as MBL2 and TGFb1 among
others (7,25,36,37). In our study we analyzed the association

Table 3. Correlation between pulmonary phenotype and genotypes of MBL2 and ADRB2 gene in 101 patients with severe CFTR mutations

Genotype of rs11003125 allelic variant rs5030737 rs1042713

Variable GG (n [ 22) GC (n [ 64) CC (n [ 15) p p p

Gender (% female) 54.5% (12/22) 54.7% (35/64) 40% (6/15) 0.577a 0.826a 0.383a
First isolation (% P. aeruginosa) 45.5% (10/22) 56.2% (36/64) 53.3% (8/15) 0.059a 0.287a 0.881a

Mean  SDb

Age at onset of clinical manifestations (years) 1.0  1.5 0.7  1.2 1.7  2.5 0.657 0.294 0.788
Age at diagnosis (years) 3.9  3.3 3.0  3.5 7.0  5.1 0.119 0.545 0.219
Age of first P. aeruginosa isolation (years) 5.8  3.2 4.3  3.7 8.7  4.2 0.010 0.294 0.219
Age of first spirometry (years) 7.8  1.3 8.9  4.7 10.7  3.6 0.003 0.898 0.019
Annual decline of FEV1 (%/year) 6.6  7.6 6.0  4.1 4.5  2.3 0.581 0.347 0.810
Brasfield score 18.5  3.0 16.8  3.2 15.7  3.3 0.014 0.212 0.815
Sweat chloride 108.4  12.3 105.2  11.5 102.7  11.0 0.930 0.122 0.237
Age of death (years) (n 5 6) 10.1  7.2 (n 5 27) 11.3  7.2 (n 5 5) 18.4 11.8 0.888 1.000 0.589

c Test.
a 2
b
Kruskal-Wallis Test.
 Gene Modifiers Associated with Cystic Fibrosis
Table 4. Association test with Recessive, Co-dominant and Dominant Model; OR and p-values
Allelic Variant Dominant Model p Co-dominant Model
MBL2_rs11003125 OR 5 2.87 (1.14e7.27) 0.022
ORa 5 2.41 (0.76e7.65) 0.127
MBL2_rs5030737 OR 5 1.99 (0.94e4.24) 0.070
ORa 5 2.56 (1.02e6.40) 0.041
B2AD_rs1042713 OR 5 0.37 (0.19e0.75) 0.005
ORa 5 0.38 (0.17e0.86) 0.018
a
The Odds Ratio adjusted was done only with patients CFTR severe mutations.
between SNPs on a1-AT, a1-ACT, IL10, TNFa, MBL2,
ADRB2, NOS3, and GSTP1 genes and CF lung disease,
and we only found association in three allelic variants,
rs11003125 (MBL2-550) and rs5030737 (D allele or
p.Arg52Cys) of MBL2 gene, and the rs1042713 (p.Arg16-
Gly) in the ADRB2 gene (Table 2). S
anchez-Dom
ınguez
CN, et al. (30) analyzed the association between seven ge-
netic variants of four modifier genes in CF Mexican patients
(n 5 81) and control subjects (n 5 104), by comparing their
corresponding allelic variants and genotypic frequencies.
They found that one variant, the TNF2 allele, appeared to
be significantly associated with CF patients ( p 5 0.012,
OR 3.43, 95% CI 1.25e9.38), different to our results. This
discordance could be explained by the high heterogeneity
of the Mexican population (38). Based on this, we tried to
reduce genetic heterogeneity using only patients with severe
mutations of CFTR (Class IeIII CFTR-mutations), to have
adjusted p-values.
The MBL2 gene encodes for mannose-binding lectin
(MBL) and is considered one of the first potential modifier
gene of lung and liver disease (39,40). MBL2 protein is a
key factor in innate immunity, is secreted by the liver as part
of the acute-phase response, enhancing phagocytosis of path-
ogenic microorganisms (41), and its low levels of expression
were associated with severe lung disease (39,42,43). MBL li-
gands are expressed by a wide variety of microorganisms,
and the binding of these ligands to the MBL protein leads
to their opsonization by the serine protease, which interacts
with receptors on phagocytes, via the activation of the com-
plement system. Because MBL protein has the ability to bind
with a wide variety of pathogens, it is very important for the
eradication of persistent and recurrent infections with micro-
organisms such as P. aeruginosa (18), so variant alleles that
decrease MBL serum levels increase risk for many different
infections. Garred P, et al. (44) demonstrated that allelic var-
iants in the promoter region at -221 and -550 known as var-
iants X/Y (rs7096206) y H/L (rs11003125) respectively, as
well as the B (Gly54Asp: rs1800450), C (Gly57Glu:
rs1800451) and D (Arg52Cys: rs5030737) alleles in the en-
coding region of exon 1 of the MBL2 gene, result in low
expression levels of this protein, thereby serving as a risk
factor for patients with CF because of its influence on the
susceptibility and risk of infection (36). Haerynck F, et al.
(45) performed a study of the influence of polymorphisms
23
p Recessive Model p
OR 5 1.57 (0.50e4.91) 0.436 OR 5 0.60 (0.26e1.40) 0.234
ORa 5 1.14 (0.29e4.55) 0.850 ORa 5 0.46 (0.17e1.21) 0.109
OR 5 NA NA OR 5 NA NA
ORa 5 NA NA ORa 5 NA NA
OR 5 0.27 (0.09e0.85) 0.021 OR 5 0.43 (0.15e1.27) 0.117
ORa 5 0.34 (0.09e1.21) 0.087 ORa 5 0.55 (0.16e1.79) 0.314
in the genes of the lectin pathway, including MBL2, and
found that the variants in these genes are significantly asso-
ciated with an early onset of chronic P. aeruginosa coloniza-
tion. Some studies, like Chalmers JD, et al. and Gravina LP,
et al. (46,47) also found that MBL insufficiency is associated
with CF lung disease severity, however, there are different
studies with opposed results and conclusions (48,49). In
our study, we found that the variant MBL2-550
(rs11003125) is common in our group of patients
(Table 2), especially in the heterozygous form. It is known
that this allelic variant located at promoter region of the
MBL2 gene is associated with the regulation of its expres-
sion, so when we performed the analysis with clinical man-
ifestations we found that the patients carrying the higher risk
genotype (GG or GC) had earlier age of first P. aeruginosa
isolation and age of the first spirometry, and a lower Bras-
field score, suggesting that these patients might have low
or null protein concentrations, a fact that could not be
confirmed since we did not determine serum concentrations
of the proteins.
The ADRB2 gene encodes the b2 adrenergic receptor,
which is an integral membrane protein that acts as an
important G-protein-coupled immunoregulatory factor, is
expressed in various tissues and is a target for many b2 re-
ceptor agonists and antagonists that are frequently used for
the treatment of many conditions, and also is an important
mediator of cyclic AMP in the airway (50,51). In 2002,
B€uscher R, et al. (52) demonstrated that these b-2-
adrenoreceptors modify the CFTR activity in CF patients
with mutations that had residual function of CFTR
(p.Phe508del), even one ADRB2 polymorphism was shown
to be related with reduced lung function and a greater
decline in lung function, the same that we observed in
our study (p.Arg16Gly [rs1042713]). Marson FA, et al.
(53) studied 122 CF patients subjected to analyze mutations
in CFTR gene, polymorphisms in ADRB2 gene, along with
clinical and laboratorial characteristics of severity and they
found an association between the Arg16Gly polymorphism
with a better spirometry to FEV1 (%) and FEF25-75% clin-
ical markers and minor risk to PI, in comparison with other
genotypes. Also, they found that Arg16Gly and Gln27Glu
polymorphisms were associated with the response to the
bronchodilation, although in previous studies this associa-
tion was not found (54).
24 Yokoyama et al./ Archives of Medical Research 49 (2018) 18e26
The formation of haplotypes allows to simplify the study
of multiple variants, evidencing allele co-segregation in
block associated with certain clinical variables in patients
with CF. In our study, we did not identify any haplotype
associated with lung severity, however, reviewing the liter-
ature there are also discordant data in this regard. In 2009,
one of the genes included in our study, MBL2 gene, was
analyzed in Italian population, and reported an association
between patients with CF who presented with severe hepat-
ic phenotype and demonstrated the formation of the HYPD
haplotype with a value of p !0.05 (40). On the other hand,
in 2006, in the USA population, Choi EH, et al. (55) tried to
look for the formation of haplotypes in this same MBL2
gene related to severe pulmonary phenotype without ob-
taining positive results; although they found a significant
association of common variant alleles in SFTPA1 and
SFTPA2 genes with poor pulmonary outcomes among a to-
tal of 135 CF patients and in those with chronic PA
colonization.
In countries like ours, the application of complex and
advanced technologies, such as genome-wide association
study (GWAS) or whole-genome sequencing (WGS) where
thousands or millions of variants, of several cases and con-
trol are analyzed in a single trial (6), would help to carry
out these association studies more quickly and accuracy,
since the research for modifier genes and their association
with the clinical manifestations must be continued. Our re-
sults support the previous association studies which tried to
describe allelic variants in modifier genes, that together
with environmental factors modify the disease, so their pro-
tein products would become immediate targets for a thera-
peutic intervention.
The treatment of CF is focused to clinical manifesta-
tions; classical management includes pancreatic enzyme
replacement, respiratory physiotherapy, mucolytic therapy,
and aggressive antibiotic therapy (56,57). Cystic fibrosis
is realizing the promise of personalized medicine, since
current therapeutic strategies that target the causal CFTR
has enabled a better quality and life expectancy, but vari-
ability in response is demanding better prediction of out-
comes to improve management decisions (58). The last
ten years some investigators have led to the development
of new pharmacotherapies that can partially repair CFTR
function, targeting directly the primary defect of the disease
(59,60), such as intestinal organoids, viral vectors and non-
viral synthetic nanoparticles for delivery of CFTR mRNA,
sgRNA for genetic repair of CFTR mutation, or suppressor
tRNAs that facilitate stop codon read-through, and new in-
sights into mechanisms of airway epithelial repair (61).
However, there are also recent studies of CF modifier
genes as potential drug targets (62). In 2012, Dorfman R
(63) summarized studies with large patients’ cohort of mod-
ifier genes and CFTR, as drug targets, and he found that
because lung disease is the major factor in CF morbidity,
all modifier studies have been focusing primarily on this
phenotype. All other phenotypes, such as risk of early in-
fections, risk of diabetes or liver disease, risk of meconium
ileus at birth are considered to be secondary. Within these
studies we can mentioned Marson FA, et al. (53) who found
an association between the Arg16Gly and Gln27Glu poly-
morphisms in ADRB2 gene with CF’s severity and broncho-
dilator response; Strug LJ, et al. (64) analyzed the
association of the SLC26A9 gene with lung disease in Ca-
nadian and French CF patients with CFTR-gating mutations
and those homozygous for the common Phe508del muta-
tion, and found that a common variant in the SLC26A9 gene
may predict response to CFTR-directed therapeutics; and
also, other authors have been proposed the potential value
of MBL replacement therapy in some cystic fibrosis pa-
tients (46,49).
Conclusions
The allelic variation in the CFTR gene is consistent with
some aspects like PI, however, other characteristics such
as lung disease seem to have an independent genetic control
besides CFTR gene mutations. To date, the modifier genes
are part of this, and are still of great importance since asso-
ciation studies like ours, continue trying to determine vari-
ants in other genes than CFTR. Taking into account our
results and those that have been previously reported, we
consider that as part of the creation of the new so-called
personalized therapy in CF, whose core is the therapy
aimed at correcting CFTR gene, it would be worthwhile
to apply it, in the future not very distant, in genes such as
ADRB2 or MBL2 that modify some clinical manifestations
especially at the lung disease, since it is still the main cause
of morbidity and mortality in this group of patients. Finally,
we insist on not leaving aside this type of studies, since
having techniques such as GWAS or WES, all those inves-
tigators interested in this area of research will be able to
advance in achieving a better quality of life for CF patients
with severe lung disease.
Acknowledgments
The authors wish to thank the Asociaci
on Mexicana de Fibrosis
Qu
ıstica, CONACYT (CONACYT-SALUD 2003-C01-066), and
Federal Founds (‘‘Caracterizaci
on de genes modificadores relacio-
nados con la expresividad variable de la fibrosis qu
ıstica’’ - INP
13/2004) for their financial support.
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