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S. Concha and R. Hoyos-Bachiloglu
severe neutropenia are at risk of sepsis, periodontitis, gingivitis, and oral aphthae, whereas those with less profound
neutropenia are at risk of infections due to the Staphylococcus
and Streptococcus species [6].
Chronic Granulomatous Disease (CGD)
This is a genetically heterogeneous disease characterized by
recurrent life-threatening bacterial and fungal infections and
dysregulated granuloma formation caused by the absence,
low expression, or malfunction of one of the phagocyte nicotinamide adenine dinucleotide phosphate hydrogen
(NADPH) oxidase components, resulting in a defect of the
phagocyte respiratory burst and the generation of phagocyte
superoxide used to destroy microbes [29].
The clinical manifestations of this disease depend on the
underlying genetic defect. Most patients with the X-linked
form present infections early in life and have a more severe
clinical course and have an increased mortality compared to
patients with the autosomal-recessive disease. These individuals are most affected by infections due to catalasepositive bacteria such as Staphylococcus aureus,
Burkholderia cepacia, and Klebsiella, Aerobacter, and
Serratia spp. Fungal infections are also extremely common
in these patients, mostly due to Aspergillus. The most
infected sites in CGD are the skin, soft tissue, bone, sinus,
gut, and lungs, although complicated acute otitis media has
been reported as the rst sign of the disease [30].
The diagnosis of CGD is made by an absent or signicantly decreased respiratory burst in stimulated neutrophils
by means of a dihydrorhodamine (DHR)-123 test. This test
measures the production of superoxide by phagocytes after
stimulation with phorbol myristate acetate (PMA) by ow
cytometry using uorescent probes like DHR-123. In stimulated neutrophils, DHR is oxidized by hydrogen peroxide
(produced in the presence of normal NADPH oxidase and
myeloperoxidase) to rhodamine-123 that emits uorescence
[31].
Treatment is based on the management of infections and
on the use of antibiotic and antifungal prophylaxis, with or
without the use of interferon-gamma. The only known cure
for this disease is hematopoietic stem cell transplantation
(HSCT) [32].
Combined Immunodeciencies (CIDs)
Combined T- and B-cell immunodeciencies are also associated with otitis media, although patients affected by these
conditions usually present with opportunistic infections and
recurrent bacterial, viral, fungal, and parasitic infections.
Combined immunodeciencies with defective control of
viral infections may have an increased risk of malignancy,
especially Epstein–Barr virus lymphoma, and can present
autoimmune disease, allergy, and autoinammation. They
can also have nonimmune manifestations and manifest with
a syndromic presentation [11].
Severe combined immunodeciencies are characterized
by profound impairment of T-cell development and/or function, so children will present in the rst months of life with
severe opportunistic infections, and, in the absence of denitive therapy, patients inevitably die within the rst year of
life [33].
Other less profound combined immunodeciencies have
also been associated with recurrent sinopulmonary infections. CD40 ligand deciency, also known as X-linked
hyper-IgM (X-HIGM) syndrome, results in the absence or
non-function of molecules involved in immunoglobulin class
switch recombination, a critical process in the production
and maturation of antibodies triggered by the T cell–B cell
interaction. Patients with hyper-IgM syndrome present with
infections early in life, most frequently pneumonia (86%)
and upper respiratory tract infections like otitis media and
tonsillitis (70%). They can also present with neutropenia and
gastrointestinal manifestations like sclerosing cholangitis
[34]. Laboratory tests show normal or elevated IgM levels
associated with low IgG and IgA in most patients. Diagnosis
is made by CD40L expression on activated CD4+ T cells by
ow cytometry and genetic test. Treatment usually includes
IVIG replacement and antimicrobial prophylaxis; however,
the only curative treatment is HSCT [35].
Combined immunodeciencies with associated or syndromic features can also present recurrent otitis media. Such
conditions include Wiskott–Aldrich syndrome, ataxiatelangiectasia, chromosome 22q11.2 deletion syndrome, and
hyper-IgE syndrome.
Children with Wiskott–Aldrich syndrome usually present
with thrombocytopenia with small platelets, eczema, bloody
diarrhea, and bacterial infections. They experience a progressive decline in T cell number and low IgM and low antibody responses to polysaccharides [36].
Ataxia-telangiectasia presents with decreasing T cell
numbers, associated with low IgG and IgA, increased alphafetoprotein, increased radiosensitivity, and malignancies due
to chromosomal instability [37].
Patients with thymic defects like chromosome 22q11.2
deletion syndrome or DiGeorge/velocardiofacial syndrome
present with hypoparathyroidism, conotruncal cardiac malformations, and thymic defect determined either by a small
or absent thymus and low T-cell counts. They frequently
present with otitis media mainly due to anatomical defects of
the upper respiratory tract like palatal anomalies [38].
Hyper-IgE syndromes are an heterogeneous CID group of
different genetic defects like STAT3 (signal transducer and
activator of transcription 3) deciency and DOCK8 (dedicator of cytokinesis 8) deciency that manifest as eczema, high
IgE, infections like recurrent ear and sinopulmonary infec-
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87
tions, skin abscesses due to Staphylococcus, mucocutaneous
candidiasis, cutaneous viral infections, atopy, and malignancies [39].
Part IV: How toEvaluate IEI inRecurrent
Otitis Media Cases
An accurate and prompt diagnosis of patients with IEI is
critical for timely initiation of therapeutic measures and to
provide the patients and their families with appropriate
genetic counseling. Given the diversity of IEI that can present with recurrent ear and sinopulmonary infections, laboratory evaluation must be rational and guided by the patient’s
infection history.
The nature of pathogens causing the ear infection provides relevant diagnostic information (Table9.5). Increased
susceptibility to pyogenic encapsulated bacteria such as
Streptococcus pneumoniae and Haemophilus inuenzae is
seen in defects of the antibody and/or complement system.
Infections due to a virus like Cytomegalovirus and fungi like
Candida and Pneumocystis jirovecii are frequent in patients
with T-cell defects. Patients with Staphylococcus aureus,
Serratia marcescens, Burkholderia cepacian, and Aspergillus
infections must be screened for phagocytic defects [9].
As IEI can alter not only the number of its components
but also their functionality, it is important not only to perform quantitative laboratory tests but also qualitative tests
(Table9.6).
When a humoral defect is suspected, immunoglobulin
levels must be evaluated and testing results compared to ageadjusted reference values. Hypogammaglobulinemia is
dened as a value below two standard deviations for age, and
agammaglobulinemia is dened as an IgG level less than
100mg/dL.Functional evaluation of the humoral response
can be done by evaluating serum titers to common vaccines,
protein-based vaccines such as tetanus and diphtheria, and
polysaccharide-based vaccines such as pneumococcus. If
serum vaccination titers are below normal, revaccination and
assessment of titers 4–6weeks later are warranted.
When a B- or T-cell defect is suspected, a complete blood
count study should be requested to evaluate the number of
lymphocytes and lymphocyte subsets by ow cytometry to
enumerate them. It can be observed that lymphocyte count
varies with age; in newborns, the normal value is considered
over 2.0×103/mm3, but, in adults, a normal value is over
1.0×103/mm3 [40]. Functional tests that evaluate the T-cell
compartment are meant to quantify their ability to proliferate
in response to different stimuli; this can be done using nonspecic agents known as mitogens or specic antigens [41].
The evaluation of patients with suspected phagocytic
defects must include a total blood count to measure neutrophil count and chemotaxis, phagocytosis, or respiratory burst
testing to evaluate their functionality.
Table 9.6 Laboratory tests for the qualitative and quantitative study of
inborn errors of immunity
Immunity Quantitative Qualitative
Humoral Total
immunoglobulin
count
Cellular Total blood count
(lymphocytes)
Lymphocyte subsets
Phagocyte Total blood count
(neutrophils)
Complement C2, C3, C4, etc. CH50, AH50
Specic antibody production
(pneumococcus, tetanus)
Lymphoproliferation
Chemotaxis, phagocytosis,
respiratory burst
Table 9.5 Pathogens associated with the underlying inborn errors of immunity
Agent Antibody defects Combined defects Phagocytic defects Complement defects
Viruses Enterovirus Cytomegalovirus
Bacteria S. pneumoniae
H. inuenzae
M. catarrhalis
P. aeruginosa
S. aureus
N. meningitidis
Mycoplasma
Mycobacteria No Non-tuberculous, including Bacillus Calmette-
Fungi No Candida species
Protozoa Giardia lamblia P. jirovecii
Epstein–Barr virus
S. pneumoniae
H. inuenzae
M. catarrhalis
P. aeruginosa
S. aureus
N. meningitidis Mycoplasma
S. typhi
L. monocytogenes
Guérin (BCG)
Aspergillus species
Cryptococcus neoformans
Histoplasma capsulatum
Toxoplasma gondii
Cryptosporidium
No No
S. aureus
P. aeruginosa
S. typhi Nocardia
Non-tuberculous,
including BCG
Candida sp., Aspergillus
sp.
No No
S. pneumoniae
H. inuenzae
M. catarrhalis
P. aeruginosa
S. aureus
N. meningitidis
Mycoplasma
No
No
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S. Concha and R. Hoyos-Bachiloglu
Evaluation of a patient with a suspected defect in the
complement pathway should start with a functional evaluation of the classic pathway by CH50. This test can be coupled with an AH50 to complete the functional evaluation of
the complement system. The next step in the evaluation of
such patients consists of the quantication of different complement components based on functional testing results,
most commonly by nephelometry or enzyme-linked immunoassay (ELISA) variations [20]. Patients with C1, C2, or C4
deciency will have a low CH50 but normal AH50. Patients
with a low AH50 but normal CH50 have a deciency of factor B, factor D, or properdin. A decrease in both CH50 and
AH50 suggests a deciency in a shared complement component, C3 or C5–C9 [41].
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Genetics andOtitis Media
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NamK.Lee andRegieLynP.Santos-Cortez
10
Introduction
Otitis media (OM) is a complex disease with various risk
factors for its development, chronicity, and recurrence as
well as a multifactorial etiology that is yet to be fully understood. An intricate interplay among environmental, genetic,
microbial, anatomic, and immunological factors contributes
to the pathogenesis of OM.Within this context, young age,
lack of breastfeeding, use of paciers, day-care attendance or
overcrowding at home, and exposure to tobacco smoke and
particulate matter, to name a few, have been identied as
environmental risk factors for the development of OM
[1–3].
Within the otolaryngology practice, the presence of family history remains one of the most important inquiries made
during an evaluation of a child with OM.Inherited or genetic
factors were found to inuence the early onset and development of OM, with a higher incidence observed in certain
populations [4–7]. For example, the frequency of OM is
increased in children with chromosomal abnormalities such
as Down syndrome (MIM 190685) and Turner syndrome
(MIM 300082), in which the affected chromosomes often
contain numerous genes of various functions [8, 9].
Chromosome 21, which is affected in Down syndrome, contains many genes that are involved in the immune system,
particularly those that play a role in interferon signaling,
thereby causing a predisposition to viral and bacterial infections [10–15]. Compounded by distinct craniofacial structures of Down syndrome and its associated dysfunctions,
such as small middle ear compartments, poor palatal tone,
and maldeveloped Eustachian tubes, individuals with Down
syndrome often experience OM that does not resolve easily,
requiring multiple tympanostomy tube procedures, and sub-
N. K. Lee · R. L. P. Santos-Cortez (*)
Department of Otolaryngology-Head and Neck Surgery, School of
Medicine, University of Colorado Anschutz Medical Campus,
Aurora, CO, USA
e-mail: nam.lee@cuanschutz.edu;
regie.santos-cortez@cuanschutz.edu
sequent persistent tympanic membrane perforation and hearing loss [16–19]. Turner syndrome is similar in its clinical
manifestation with immune alterations found in T cells and
immunoglobulins (Igs) along with craniofacial abnormalities
of the palate, jaw, and ears that range from the external to
inner ear system [20–22]. As a result, affected individuals are
predisposed to a range of otologic problems from recurrent
or chronic OM to conductive or sensorineural hearing loss
and tympanic membrane pathology [9, 23].
The socioeconomic impact of health-care costs and delay
in speech and language for OM-affected individuals underline the need for further research in OM.Although our current understanding of the main contributors of genetic
susceptibility to OM remains scant compared to our overall
genetic knowledge of other common diseases, such as cardiovascular disorders and cancer, continued efforts toward
the identication of the determinants of human susceptibility
and host genomic responses to OM will help elucidate its
multifactorial etiology. Recent advances in genomic databases, sequencing, and analytic techniques will benet the
ongoing genetic investigation into OM and ultimately lead to
a better understanding of its pathogenesis, which, in turn,
can improve strategies for its prevention and treatment.
Human DNA Studies
Heritability
Heritability, in the narrow sense, is an estimate of the proportion of the variance in phenotypic or trait values under study
(in this case, OM), which is due to genetic factors [24].
Because OM is a complex trait, heritability has been estimated using data from related individuals as was done in
twin studies performed in three cohorts from Norway, the
United States, and the United Kingdom (UK). The initial
twin study from Norway in 1997 consisted of 2850 twins,
which was then followed up in 2004 with an enlarged cohort
of 4247 twins [25, 26]. In the initial study, gender differences
© The Author(s), under exclusive license to Springer Nature Switzerland AG 2023
M. V. Goycoolea et al. (eds.), Textbook of Otitis Media, https://doi.org/10.1007/978-3-031-40949-3_10
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N. K. Lee and R. L. P. Santos-Cortez
were detected in female twins who had higher heritability for
OM at 74% compared to males with 45% heritability [25]. In
the follow-up study that included an additional 1397 twins,
gender-based differences for heritability estimates were not
observed, although the overall heritability for OM remained
high at 61–72% [26].
In Pittsburgh, a prospective cohort study of 168 same-sex
twins and 7 same-sex triplets was conducted to determine the
heritability of time or duration of middle ear effusion (MEE)
[27]. The same cohort was reassessed after 5years, and it
was determined that the heritability of the time with MEE as
a phenotype remained consistent [28]. Heritability was 73%
in the initial study and 72% in the follow-up study, and both
had p<0.001 [27, 28].
Rovers etal. (2002) assessed the heritability of both OM
and chronic airway blockage in a prospective study of 1373
twins from the UK [29]. The heritability of acute OM was
estimated to be 57%. For both OM and chronic airway blockade, an increasing genetic contribution to the phenotypic
variance was observed at ages 2 years (49%), 3 years (66%),
and 4 years (71%). However, no gender differences were
observed [29].
Valuable information can also be gleaned from familial
studies with multiple OM-affected siblings as in the study by
Hafrén [30]. Heritability estimates of recurrent acute and
chronic OM were determined in their survey of 2436 children, who underwent surgery for OM, and their relatives
from 590 Finnish families. Chronic OM had lower heritability (22%) compared to recurrent acute OM (38%), and the
overall heritability of all types of OM was estimated at 48%
[30]. As expected, heritability estimates are higher in identical twins than in other familial relations due to the greater
similarity in both genetic makeup and environmental background of twins. Nonetheless, these heritability studies,
albeit with various heritability estimates, phenotypic denitions, ethnic background, and cohort composition, conrm
the signicant contribution of genetic factors to OM
susceptibility.
Genetic Linkage Studies
Genetic linkage studies test whether a pattern of inheritance
of a disease or trait coincides with the genotypes of families:
genetic linkage is therefore a test of co-segregation of a variant or variants inherited together (e.g., a haplotype) with the
known disease status of family members. The main metric
for linkage is the logarithm of odds (LOD) score, which is
usually considered statistically signicant if a LOD score of
3.3 or greater is obtained for a variant or haplotype within
pedigrees that co-segregate a disease or a trait [31]. To obtain
a signicant LOD score, either a large enough pedigree or a
consanguineous family with multiple affected individuals is
needed. Alternatively, smaller families that each on its own
cannot obtain linkage but, when analyzed together, can lead
to mapping of signicant loci may be used. Before the advent
of next-generation sequencing, genetic variants that are distributed across the genome were genotyped and then tested
for linkage in order to identify a genomic region where the
pathogenic variant is statistically the most likely to occur.
Early linkage studies using different cohorts of families
identied multiple loci in various regions of the human
genome, most signicantly on chromosome 10q (Table10.1).
It should be noted, however, that in many of the mapped loci,
Table 10.1 Genomic loci mapped for otitis media susceptibility in linkage studies using family data
Loci Studies LOD score Signicance and candidate genes within the loci
19q13.42–q13.43 Daly etal. [32]
Chen etal. [35]
10q26.3 Daly etal. [32]
Rye etal. [33]
LOD 2.61 (p=5.3×10−4)
LOD 3.75 (p=1.6×10−5)
LOD 3.78 (p=3.0×10−5)
Zlr 2.69 (p=3.6×10−3)
Leukocyte receptor cluster (LRC)—cluster of genes
with polymorphic functions; mainly prevents
immune cell activation [38]
Zinc nger and zinc nger-related genes (ZNF71,
ZNF8, ANF304)
Inammasome protein complex (NLRP13, NLRP5,
NLRP8)
ADAM8—identied in allergen-induced asthma,
expressed in leukocytes [193, 194]
ADAM12—involved in the epidermal growth factor
receptor signaling pathway [41, 194]
Dedicator of cytokinesis 1 (DOCK1)—phagocytosis
of apoptotic cells, skeletal and respiratory muscle
tissue development in embryogenesis [42]
TCERG1L and PPP2R2D—an intergenic region
Transcription elongation regulator-like protein
(TCERG1L)—suggested in fasting insulin, attention
decit disorder
Protein phosphatase 2A (PPP2R2D)—a modulator
of the TGF-β/Activin/Nodal pathway [33, 43]
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Table 10.1 (continued)
Loci Studies LOD score Signicance and candidate genes within the loci
3p25.3 Daly etal. [32] Unconditional:
17q12 Casselbrant etal. [197]
10q22.3 Casselbrant etal. [197]
Rye etal. [33] Z
7q33 Casselbrant etal. [197]
6p25.1 Casselbrant etal. [197]
4p15.2 Casselbrant etal. [197]
15q26.1 Allen etal. [59]
5p15.33 Allen etal. [59] p=0.045
2q31.1 Allen etal. [59]
LOD 0.60
Conditional:
10q LOD 2.43
19q LOD 1.84
LOD 2.83 (p=7.0x10
LOD>2 (p=1.8×10
1.64 (p=0.05) –
lr
LOD>2 (p=1.0×10
LOD>2 (p=2.6×10
LOD>2 (p=3.0×10
p=9.1×10
p=1.3×10
−7
−5
−5
)
−3
)
−3
)
−3
)
−3
)
Presumed to be involved in the gene–gene
interaction between 19q13.42 and 10q26.3
HRH1—an inammatory mediator [195]
IRAK2—mediates inammatory gene expression
via NF-κB [196]
Adaptor-related protein complex2, beta 1 subunit
(AP2B1)—CD8
C–C motif ligand (CCL)—recruitment of
eosinophils [199]
SFTPA2—part of surfactant protein A, which
regulates the phagocytosis of pathogens; expressed
in the Eustachian tube [45]
–
–
–
KIF7—the region is related to the splice site;
regulates sonic hedgehog [200] and Indian
hedgehog [64] via protein trafcking
TICRR—initiation of DNA replication [65]
Tubulin polymerization- promoting protein
(TPPP)—located at the intron; affects microtubule
function [63]
Non-coding region: possible role in the regulation
of LDLR (ch19), which is expressed in ciliated
airway epithelial cells [201]
+
downregulation [198]Chemokine
93
the pathogenic variants responsible for OM susceptibility
remain unknown. This is partly due to the lack of genetic
studies performed in families with OM and potentially due to
genetic heterogeneity for OM where families carry variants
within multiple genes that are mostly rare or private variants,
such that current family cohorts lack the power to detect
signicant OM loci. Below is a review of loci that have been
mapped using families with OM.
19q13.42–q13.43
Daly etal. (2004) rst identied the 19q13.42–q13.43 region
as a suggestive susceptibility locus (LOD 2.61, p=5.3×10−4)
using genotype data from Minnesotan families with OM
[32]. Approximately 100 annotated genes are present in this
region, but many of their functions remain unknown. The
leukocyte receptor cluster (LRC) genes were suggested as
potential candidate genes within the 10q locus. Within this
region are genes encoding the leukocyte Ig-like receptor
(LIR; also known as Ig-like transcripts) and the killer cell
immunoglobulin-like receptor (KIR), which are transmembrane proteins expressed on cells of immune function for
various protein activation or inhibition [33]. Such proteins
include tyrosine phosphatases Src homology region 2
domain-containing phosphatase (SHP)-1 and/or SHP-2 [34].
This region was replicated by Chen etal. (2011), who nemapped the locus to chromosome 19q13.43 using additional
single nucleotide variant genotypes (LOD 3.75,
p=1.6×10−5) [35]. The additional candidate genes identi-
ed within the 19q locus include genes related to zinc ngers, the tumor necrosis factor-alpha (TNFα), bone
morphogenetic protein (BMP), and broblast growth factorbeta (FGFβ) pathways, lymphocyte activation, and the
inammasome protein complex, all of which regulate innate
immunity in response to harmful stimuli [35–37].
10q26.3
In an initial linkage study for OM using the Minnesota family cohort, the 10q26.3 locus was identied with a LOD
score of 3.78 (p=3.0×10−5) and was replicated in another
cohort of Western Australia-based trios by Rye etal. in 2014
(Zlr 2.69, p=3.6×10−3) [32, 33]. Herein, trios are composed
of the OM-affected individual (proband) and both parents,
without affected or unaffected siblings. Within the 10q26.3
locus, the Minnesotan and Australian studies identied candidate genes encoding a disintegrin and metalloproteinase
(ADAM) domain. ADAM8 is found in leukocytes in response
to allergen exposure in asthma and is also upregulated in epithelial cells of the airway during allergic inammation [38,
39]. The function of ADAM12 is most strongly associated
with cell adhesion and fusion, extracellular matrix restructuring, and cell signaling [40]. It has been found to be upregulated in the middle ear in response to tobacco smoke [41].
Other genes implicated in this region include DOCK1 (MIM
601403), which is involved in phagocytosis; TCERG1L
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N. K. Lee and R. L. P. Santos-Cortez
encoding transcription elongation regulator-like protein, and
PPP2R2D (MIM 613992), which modulates the modulator
of the transforming growth factor (TGF)/Activin/Nodal
pathway [33, 42, 43].
10q22.3
Chromosomal region 10q22.3 has also been identied (LOD >2,
p = 1.81× 10−3) and replicated with suggestive evidence (Zlr
1.64, p=0.05), though not reaching statistical signicance [2,
33]. SFTPA1 (MIM 178630) and SFTPA2 (MIM 178642) are
genes within this region that together encode the human surfactant protein (SP-A) and have previously been implicated in OM
[44]. SP-A haplotype and genotype variations have been
observed in children with the rst episode of OM before
6months, and common variants in these genes were associated
with protection against OM in infants at risk of asthma [45, 46].
SP-A is expressed in the middle ear mucosa and in the Eustachian
tube and is known to contribute to innate immune responses by
increasing the phagocytosis of otopathogens [45–48].
Candidate Gene Association Studies
Using a gene of interest, candidate gene association studies
estimate the frequency with which the minor allele, or the less
prevalent allele in the background population, is found in
OM-affected patients in comparison to a control group. Healthy
or unaffected individuals that are related to the OM-affected
patients within the same families, or unrelated healthy individuals with no previous history of OM, may be used as controls
(case–control study). Numerous studies have been conducted,
particularly using candidate genes, relating to inammation
and innate immune responses, but many candidate gene studies
will not lead to a signicant association when a more stringent
threshold for genome-wide signicance is applied. Nonetheless,
some genes that were initially identied in smaller case– control
association studies, such as the HLA and ABO (MIM 110300)
genes, were also deemed signicant loci in genome-wide association studies (GWASs) for OM [49–56].
Genome-Wide Association Studies (GWASs)
A GWAS is currently the starting point in identifying the loci
of interest. It assesses the association of variants spread
throughout the genome with the trait or disease under investigation and, in contrast to candidate gene association studies, does not assume an association between a locus of
interest and the trait. Instead, a GWAS employs an agnostic
approach to identifying the loci of interest, which may be a
regulatory variant often found in the non-coding region, a
haplotype encompassing variants that are inherited together,
or a gene harboring multiple rare variants associated with the
trait. For OM, a series of GWASs have been conducted using
common variant genotypes from microarrays, with each
describing independent ndings (Table10.2). With advances
Table 10.2 Otitis media susceptibility genes identied from genome-wide association studies
Study SNP Cases Controls Cohort Signicant associations
Rye etal. 2012
[33]
Allen etal. 2013
[59]
Einarsdottir etal.
2016 [66]
van Ingen etal.
2016 [70]
2,524,817 416 1075 Western Australian
Pregnancy Cohort (Raine)
Study
324,748 373 229 University of Minnesota
[20]
University of Pittsburgh
[24]
964,193 829 2118 Finnish rs16974263 (p=1.8×10−7)
460,000 825 7936 European-descent
Americans
rs6755194 (p = 8.3×10−7)
rs1862981 (p = 2.2×10−5)
rs1110060 (p=9.1×10−7)
rs10497394 (p=1.5×10−8)
rs10775247 (p=6.3×10−5)
rs268662 (p=1.6×10−6)
rs4150992 (p=3.4×10−6)
rs2932989 (p=4.4×10−8)
rs3767498 (p=1.25×10−6)
rs12725646 (p=4.3×10−5)
rs255142 (p=1.9×10−6)
rs9514552 (p=2.1×10−5)
rs12888576 (p=7.8×10−6)
rs2809139 (p=3.2×10−6)
rs8036951 (p=3.8×10−5)
rs10409140 (p=4.4×10−5)
a
Candidate genes
CAPN14
GALNT14
BPIFA gene cluster
KIF7
TICRR
TPPP
PLD3
SERTAD1
SERTAD3
HIPK4
PRX
BLVRB
FNDC1
KIF21B
CACNA1S
ASCL5
Intergenic region near
MIR205HG
CRHR2
INMT
ARGLU1
BDKRB2
Intergenic region near
C14orf177
FAM189A1
TPM4
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Table 10.2
Study SNP Cases Controls Cohort Signicant associations
Pickrell etal.
2016 [49]; Tian
etal. 2017 [53]
a
p-values in bold font indicate those that passed the genome-wide signicant thresholds of the study
in computational efciency, continuous decline in the cost of
sequencing, and increasing availability of large-scale biobank data with genotypes and OM phenotypes, the discovery
of additional novel ndings from GWASs, which include
both common and rare variants, is possible in the near future.
from Australian trios and identied three novel candidate
genes/gene clusters, namely, (1) rs6755194 within chromosome 2p23.1, which is either upstream of or intronic to two
isoforms of CAPN14 (MIM 610229); (2) rs1862981, also
located on 2p23.1, intronic to GALNT14 (MIM 60822) and
proximal to CAPN14; and (3) BPIFA clusters, including
BPIFA1 (MIM 607412), BPIFA2, and BPIFA3 on the
genomic region 20q11.21 [57]. However, these ndings
could not be replicated in an independent cohort [58]. Other
genes identied in this study with trends toward association
were linked to the TGF-β pathway [57].
tied an intergenic locus rs10497394 between the genes
CDCA7 (MIM 609937) and SP3 (MIM 601804) on 2q31.1,
which was subsequently replicated in an independent
Pittsburgh-based US cohort as being associated with both
chronic OM with effusion and recurrent acute OM [59].
This locus was found to regulate the expression of LDLR
(MIM 606945) on chromosome 19, a gene that is expressed
in ciliated epithelial cells of the airway with its transcribed
protein predicted to be a binding site for human rhinovirus
C, a known pathogen for upper respiratory tract infections
and OM [60]. Newer data from the Genotype-Tissue
Expression (GTEx) database showed that the rs10497394
variant signicantly regulates RNA levels of CDCA7 in
thyroid and arterial tissues; however, GTEx does not
include data from middle ear tissues [61]. CDCA7 mutations cause immunodeciency, centromeric instability, and
facial anomalies syndrome (MIM 616910), wherein hypoor agammaglobulinemia leads to recurrent life-threatening
(continued)
560,000–
950,000
46,936 74,874 European-descent
Americans
In the rst GWAS on OM, Rye et al. (2012) used data
In the Minnesota-based cohort, Allen etal. (2013) iden-
rs681343 (p=3.5×10
rs1978060 (p=1.2×10
rs2808290 (p=5.1×10
rs7174062 (p=3.5×10
rs4329147 (p=9.6×10
rs8176643 (p=3.7×10
rs1802575 (p=1.5×10
rs5829676 (p=1.8×10
rs72931768 (p=2.6×10
rs35213789 (p=3.8×10
rs114947103 (p=5.4×10
rs13281988 (p=9.8×10
rs67035515 (p=1.6×10
rs73015965 (p=3.8×10
infections [62]. Additional loci on chromosomes 5 and 15
(rs386057, rs1110060, and rs10775247) were also identied but did not reach genome- wide signicance in the replication study [59]. The genes affected by these three
variants are involved in microtubule function, regulation of
mammalian sonic hedgehog and Indian hedgehog pathways, and DNA replication [63–65].
Einarsdottir etal. (2016) found three variants, all on chromosome 19, to be associated with childhood OM in the
Finnish population [66]. None of the identied regions overlapped with those previously found in association with OM
on chromosome 19. Genome-wide signicance was established for rs16974263, a variant intronic to the PRX (MIM
605725) gene, which was found in association with chronic
OM with effusion in a UK cohort, albeit with opposite directions of effect [66]. Out of the several genes identied within
this region are three candidate genes, PLD3 (MIM 615698),
SERTAD1 (MIM 617850), and BLVRB (MIM 600941),
which are previously known to be associated with immune
function with expression found in macrophages [67, 68]. In
the GTEx database, the rs16974263 variant regulates either
the RNA levels or splicing of isoforms for SERTAD3, HIPK4
(MIM 611712), PLD3, and PRX in various tissues [61]. In
particular, SERTAD3 is expressed in the mucosal tissue and
its protein inhibits the replication of inuenza A virus upon
induction by type I interferon responses during an infection
[61, 69].
Additional studies in Americans of European-descent
identied genome-wide signicance for a variant on chromosome 6 (rs2932989) that alters the methylation status of
the gene encoding bronectin type III domain containing 1
(FNDC1, MIM 609991) [70]. Although its function is not
clearly elucidated, the study found an upregulation of this
gene in the middle ear tissue under pro-inammatory conditions [70]. Other candidate genes from suggestive loci largely
contained those related to immune responses.
a
−30
Candidate genes
)
HLA genes
−19
)
Several intergenic regions
−16
FUT2
)
−14
ABO
)
−12
TBX1
)
−11
MKX
)
−10
AUTS2
)
−10
CDHR3
)
−9
PLG
)
−9
)
−9
)
−9
)
−8
)
–8
)
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N. K. Lee and R. L. P. Santos-Cortez
Finally, 14 genome-wide signicant loci were identied by
Pickrell etal. (2016) and Tian etal. (2017) using GWAS data
from 23&Me [49, 53]. Among these loci, the OM-associated
genes included FUT2 (MIM 182100) and ABO, both involved
in glycosphingolipid biosynthesis, as well as TBX1 (MIM
602054) and MKX (MIM 601332), which encode transcription factors. Genes important for embryogenesis and neurodevelopment (FGF3 (MIM 164950) and AUTS2 (MIM 607270)),
as well as those implicated in asthma (CDHR3 (MIM
615610)), and spontaneous chronic OM in mice (PLG (MIM
173350)) were also found in association with OM [71–73]. To
date, only 4 of these 14 loci, FUT2, TBX1, ABO, and CDHR3,
have been replicated in independent human cohorts with additional evidence from functional or multi- omics studies performed for FUT2 and CDHR3 [49–52, 74–78].
To identify pathways that are potentially important for
OM susceptibility, 21 genes with p<5×10−5 available in the
literature were selected from candidate gene association
studies and GWAS results. Using NetworkAnalyst software
[79–81], these genes were then used as an input for dening
protein–protein interaction networks. Within these networks
exist subnetworks that are involved in signicant pathways,
highlighting the underlying pathophysiology and serving as
potential targets for novel therapeutics (Fig.10.1). The major
pathways identied included a rhythmic process, a viral pro-
Fig. 10.1 Protein–protein interactions in the network analysis of genes
implicated in otitis media from exome sequencing and genome-wide
association studies. The green nodes represent proteins encoded by
input or seed genes with a p<5×10−5 from exome sequencing and
genome-wide association studies. The orange nodes represent proteins
that are found in association with the seed proteins within a signicant
cellular process
cess, negative regulation of an? apoptotic process, transcription, transcription by RNA polymerase II, chromatin
remodeling, regulation of cell cycle, and circadian rhythm
(Table 10.3). These identied pathways suggest that basic
Table 10.3 Signicant pathways identied from network analysis of
genes implicated in otitis media from exome sequencing and genomewide association studies
Signicant pathways p-value Genes
Rhythmic process
Viral process
Negative regulation of the
apoptotic process
7.9×10
1.99×10
2.95×10
−17
−16
−13
PPARG
NCOA2
CREBBP
TP53
PRKDC
UBE3A
PML
EP300
NRIP1
PPARGC1A
SP1
MAGED1
MDM2
YWHAE
COPS6
KAT5
CREBBP
TP53
STAT3
CD4
TSG101
NEDD4L
UBE3A
PML
HNRNPA1
EP300
FYN
SP1
STAT1
IRAK1
RELA
MAP3K7
MMP1
ROR2
MDM2
AKT1
EGFR
YWHAZ
PTK2B
STAT5B
IL6
TMF1
TP53
RAF1
PRKDC
CD38
STAT3
SMAD3
NFKB1
MED1
IRAK1
RELA
IL10
UBC
COPS5
TOP1
CREB1
DDX5
SFPQ
KDM5A
GNB2L1
NFKB2
HNRNPU
MLL
HDAC1
HDAC2
THRAP3
LCK
RB1
HSPD1
TOP1
SRC
CREB1
TBP
YWHAB
CEBPA
RAN
RBX1
SUMO1
GNB2L1
SET
PAK2
TRIM28
CUL1
CUL2
EIF4A2
DAXX
IKBKG
ADA
WNT1
JUN
HSPA1A
HSP90AB1
HSPD1
SRC
MIF
CBL
DUSP1
AKT2
MSX2
SNCA
CDKN1A
BCL6
PPID
PAK2
HDAC1
NTRK2
HDAC2
BAG1
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