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The genetics of auricular development and malformation: New

findings in model systems driving future directions for microtia
research
Timothy C. Cox
a, c, d, f,
*
, Esra D. Camci
a, d
, Siddharth Vora
a, d
, Daniela V. Luquetti
a, c
,
Eric E. Turner
b, e
a
Center for Developmental Biology & Regenerative Medicine, Seattle Children’s Research Institute, Seattle, WA, USA
b
Center for Integrative Brain Research, Seattle Children’s Research Institute, Seattle, WA, USA
c
Department of Pediatrics (Craniofacial Medicine), University of Washington, Seattle, WA, USA
d
Department of Oral Health Sciences, University of Washington, Seattle, WA, USA
e
Department of Psychiatry and Behavioral Sciences, University of Washington, Seattle, WA, USA
f
Department of Anatomy & Developmental Biology, Monash University, Clayton, Victoria, Australia
a r t i c l e i n f o
Article history:
Received 12 February 2014
Accepted 11 May 2014
Available online xxx
Keywords:
Microtia
Anotia
Auricular development
Craniofacial microsomia
OAVS
Goldenhar syndrome
Mouse models
a b s t r a c t
Microtia is a term used to describe a wide array of phenotypic presentations of the outer ear. Although
the majority of the cases are isolated in nature, much of our understanding of the causes of microtia has
been driven by the identification of genes underlying syndromic forms where the anomaly co-presents
with various other craniofacial and extra-craniofacial structural defects. In this review we discuss recent
findings in mice deficient in Hoxa2, a key regulator of branchial arch patterning, which has necessitated a
revision to the canonical model of pinna morphogenesis. The revised model will likely impact current
classification schemes for microtia and, as we argue in this review, the interpretation of the develop-
mental basis for various auricular malformations. In addition, we highlight recent studies in other
mammalian species that are providing the first clues as to possible causes of at least some isolated
anomalies and thus should now accelerate the search for the more elusive genetic contributions to the
many isolated and non-syndromic cases of microtia. These findings, together with the application of new
genome-level sequencing technologies and more thorough quantitative assessment of available mutant
mouse resources, promise an exciting future for genetic studies in microtia.
Ó 2014 Elsevier Masson SAS. All rights reserved.
1. Introduction
Microtia is a broad term that encapsulates a diverse array of
‘abnormal’ appearances of the auricle (or pinna) that involves
various malformations of the auricular components. Anotia, the
complete absence of auricular components, and polyotia, typically
seen as mirror-image auricular duplications, represent the ex-
tremes of auricular phenotypes [Hunter et al., 2009]. Ectopic
structures, known as pre-auricular tags, are also sometimes asso-
ciated with these pinna defects [Carey et al., 2006].
Microtia most commonly presents as a unilateral anomaly
(>75% of cases), with the right ear being affected in nearly 60% of
these cases [Canfield et al., 2009; Castilla and Orioli, 1986; Forrester
and Merz, 2005; González-Andrade et al., 2010; Harris et al., 1996;
Mastroiacovo et al., 1995; Nelson and Berry, 1984; Shawet al., 2004;
Suutarla et al., 2007]. Like many other structural birth defects,
microtia can be seen as part of a syndrome or as an apparently
isolated defect. Collectively, the prevalence of microtia ranges
between 0.83 and 4.34 per 10,000 births [Canfield et al., 2009;
Forrester and Merz, 2005; Harris et al., 1996; Luquetti et al., 2012;
Shaw et al., 2004; Suutarla et al., 2007]. While the common
perception is that microtia more frequently presents as an
isolated anomaly, a number of comprehensive investigations
clearly demonstrate that a significant proportion of affected
children (20e60% depending on the study) has either a
recognizable syndrome or at least one major associated anomaly
not directly related to the ear abnormality. Those presenting with
bilateral microtia are significantly more likely to have associated
anomalies [Canfield et al., 2009; Harris et al., 1996; Luquetti et al.,
* Corresponding author. Center for Developmental Biology & Regenerative
Medicine, Seattle Children’s Research Institute, 1900 Ninth Avenue, M/S C9S-5,
Seattle, WA 98101, USA. Fax: þ1 206 884 1407.
E-mail address: tccox@uw.edu (T.C. Cox).
Contents lists available at ScienceDirect
European Journal of Medical Genetics
j ournal homepage: ht t p: / / www. el sevi er. com/ l ocat e/ ej mg
http://dx.doi.org/10.1016/j.ejmg.2014.05.003
1769-7212/Ó 2014 Elsevier Masson SAS. All rights reserved.
European Journal of Medical Genetics xxx (2014) 1e8
Please cite this article in press as: Cox TC, et al., The genetics of auricular development and malformation: New findings in model systems
driving future directions for microtia research, European Journal of Medical Genetics (2014), http://dx.doi.org/10.1016/j.ejmg.2014.05.003
2012; Mastroiacovo et al., 1995; Shaw et al., 2004]. In all instances,
this percentage rises further when anomalies considered ‘minor’
are included [Luquetti et al., 2013a,b].
Although the term ‘microtia’ specifically refers to anomalies of
the auricle, more than 90% of patients with microtia experience
conductive hearing loss on the affected side [Bassila and Goldberg,
1989; Calzolari et al., 1999; Carey et al., 2006; Ishimoto et al., 2007;
Suutarla et al., 2007]. This can be because of additional structural
anomalies involving the external acoustic meatus (EAM, or
external auditory canal, EAC), the tympanic membrane (i.e.
eardrum), the middle ear ossicles or combinations of these. The
existence of additional structural anomalies is suggestive of a
broader developmental problem in most patients with microtia
and therefore it is important from a clinical perspective to
conduct a thorough and detailed evaluation. This should include
a careful assessment of the mandible and temporal region given
the proximity and/or common origin of the embryonic facial
tissue that gives rise to the external and middle ear structures
and the posterior aspects of the mandible. In line with this, it is
widely believed that isolated microtia represents the mild end of
the oculo-auriculo-vertebral (OAV) spectrum (OAVS; alternately
known as craniofacial microsomia or Goldenhar syndrome) [Heike
and Hing, 2009]. Patients receiving a diagnosis of OAVS frequently
exhibit microtia together with variable facial asymmetry, and often
cervical vertebral anomalies, although they can also have a wider
range of defects. Many of these anomalies are the same as those
found to be preferentially associated with isolated microtia
[Luquetti et al., 2012]. Other preferentially associated anomalies
include components of the phenotypic spectrum of Townese
Brocks [Kohlhase, 2012] and lacrimo-auriculo-dento-digital
(LADD; Ordonez and Tekin, 2012) syndromes and also
occasionally seen in branchio-oculo-facial syndrome (BOFS) [Lin
and Milunsky, 2011]. Thus, these associations may represent
variable presentations of these syndromes and therefore distinct
from truly isolated microtia.
Significant inroads have been made in identifying the causative
genes in syndromic forms of microtia using both traditional linkage
mapping and disease gene identification approaches, and more
recently exome sequencing strategies. These success stories have
been reviewed by Alasti and Van Camp [2009] and more recently by
Luquetti et al. [2012]. While this review will briefly revisit the
genetics of syndromic forms of microtia, it will primarily focus on
new data from studies in mice that revise our model of how the
auricles are formed and how existing human auricular
malformations might be interpreted from the perspective of the
underlying genetic program. In addition, we emphasize the need
and importance for better characterization of ear malformations
in patients as well as mice (as the primary model of human
congenital anomalies). We further highlight intriguing new
findings from non-traditional “model organisms” that provide an
argument for searching for non-coding, regulatory mutations and
copy number variations as a cause of isolated microtia and possibly
the broader OAV spectrum.
2. Revising the embryology of auricular development
Much of our understanding of auricular development has come
from early descriptive observations on the morphological changes
in the branchial (or pharyngeal) arches in human and animal em-
bryos and interpretation of malformations seen in the clinic and in
animal models. In the early 1880s, His [1882] described six
protuberances, now known as the ‘hillocks of His’, in the
branchial arches of human embryos. These hillocks or tubercles e
three in the first arch and three in the second arch e are first
identifiable during the sixth week of embryogenesis surrounding
the first branchial cleft, which is the space or groove between the
first and second arches. Growth and morphological change within
these arches occurs until a definitive auricular form is evident
between the eight and ninth week of development (Fig. 1a). Over
the ensuing two months, the auricle assumes its recognizable
‘adult-like’ form (see Fig. 1a and c).
From the assessment of various embryonic stages, His [1882,
1885] concluded that each of the early hillocks contributes to a
discrete component of the pinna through precisely timed growth
and differentiation. However, around the time of these reports,
there was conflicting evidence over the importance of the
hillocks and whether or not they derived the entire adult
auricular structure. For example, a few years before His’
descriptions, Moldenhauer (1877) reported similar hillocks,
although fewer in number, in the chick which lacks pinnae.
Schwalbe [1891], in 1891, also described the presence of branchial
tubercles in reptiles that also do not possess auricles. These and
other studies were nicely summarized in a comprehensive review
by Streeter [1922] in 1922, who himself began to question His’
model of auricular development. The simple ‘hillock’ model has
nevertheless prevailed in the literature with small modifications
over the years. In the most commonly reproduced version, the
hillocks of the second arch form the bulk of the pinna, those of
the first arch form the tragus and at least part of the root, and the
first branchial cleft persists as the EAM [Mallo, 2003]. More
consistent with the earlier findings, however, is the model
whereby tissue marked by the hillock locations contributes to
some specific parts of the auricle, while tissue caudal to the
hillocks of the second arch gives rise to the free ear fold (from
which the helix and scapha regions derive; Fig. 1a) [Porter and
Tan, 2005]. Furthermore, as summarized below, new data in mice
indicate that the first branchial cleft does not give rise to the EAM.
3. Auricular morphogenesis: branchial arch specific genetic
programs
The revised model of auricular morphogenesis (Fig. 1a) also
accommodates exciting new molecular genetic findings in mice
reported by Minoux et al. [2013]. These researchers extended
their earlier studies by further characterizing mice deficient for
Hoxa2, that presented as a model for anotia. Hoxa2 encodes a
homeobox transcription factor normally expressed throughout
branchial arch 2 mesenchyme, and mice deficient for Hoxa2 not
only lack “pinnae” but also exhibit duplication of the EAM
[Minoux et al., 2013; Santagati et al., 2005]. Mutations in the
coding region of HOXA2 have also been found in patients with
microtia phenotypes. Alasti et al. [2008] first reported a missense
mutation, resulting in substitution of a highly conserved
Glutamine for a Lysine at position 186 of HOXA2, in a
consanguineous Iranian family segregating for an autosomal-
recessive form of bilateral microtia. Brown et al. [2013]
subsequently described a family with dominantly inherited non-
syndromic bilateral microtia in which they identified a nonsense
mutation in HOXA2. The auricular features of both families were
similar, however affected individuals in the Iranian family pre-
sented with more severe microtia, abnormalities of the ear canal,
profound mixed hearing impairment, as well as partial cleft palate
[Alasti et al., 2008], similar to that seen in the Hoxa2-deficient mice
and confirming the clinical relevance of this model. These findings
are also consistent with a dosage sensitive effect of HOXA2; the
more severe phenotype in the family reported by Alasti et al.
[2008] likely being related to incomplete loss of HOXA2 function
in both alleles in the consanguineous family.
It is well established that homeotic genes, such as Hoxa2,
function as early specifiers of axial identity (the so called ‘Hox
T.C. Cox et al. / European Journal of Medical Genetics xxx (2014) 1e8 2
Please cite this article in press as: Cox TC, et al., The genetics of auricular development and malformation: New findings in model systems
driving future directions for microtia research, European Journal of Medical Genetics (2014), http://dx.doi.org/10.1016/j.ejmg.2014.05.003
code’; Alexander et al., 2009). Loss of such Hox gene function in one
segment of the body results in homeotic transformation of tissue
normally expressing that Hox gene into structures seen in the
adjacent anterior segment, creating “mirror-image duplications”.
Likewise, misexpression of Hox genes in anterior domains that do
not normally express the gene is typically capable of initiating the
molecular program of the more posterior structures, thus
transforming that tissue into a mirror-image duplication of the
posterior segment. To investigate this possibility in the case of
Hoxa2, Minoux et al. produced mice that ectopically expressed
Hoxa2 in neural crest-derived mesenchyme of branchial arch 1,
where it is not normally expressed. These mice presented with
striking mirror-image auricular duplications (Fig. 2aec), supporting
the conclusion that Hoxa2 specifies branchial arch 2 identity. These
mice therefore provide a valuable resource for investigating the
genetic program specifying the identity of second arch-derived
pinna structures.
A fascinating corollary from the mouse studies of Minoux et al.
was that the EAM is not derived from the first branchial cleft, as
presumed in current models of auricular development. These in-
vestigators found that in addition to the absence of auricles, Hoxa2-
deficient mice exhibited duplication of the EAM as well as the
Fig. 2. Mirror-image auricular duplications: a role for ectopic expression of the HOXA2 genetic program? The Hoxa2-regulated genetic program is normally restricted to branchial
arch 2-derived mesenchyme where it is required for normal pinna morphogenesis (a) E18.5 wildtype embryo. (b) Ectopic expression of Hoxa2 in all neural crest-derived
mesenchyme, including that contributing to branchial arch 1 structures, produces embryos with mirror-image duplications of auricular structures (black arrow) (c) higher
magnification image of a mutant embryo showing duplicated auricular structures as well as small ectopic structures reminiscent of pre-auricular tags (white arrowhead). (d) Partial
mirror-image duplication of auricular structures in a patient. Images in aec (albeit flipped in orientation from their original presentation) are reproduced from Minoux et al. [2013]
with permission from the journal Development. http://dx.doi.org/10.1242/dev.098046.
Fig. 1. Development of auricular form in humans and mice. (a) updated model of auricular morphogenesis adapted from Porter and Tan [2005], the original sketches from Streeter
[1922], and modified to accommodate the data in mice from Minoux et al. [2013]. (I) The branchial arches contributing to auricular development e the mandibular process of
branchial arch 1 (dark pink) and arch 2 (orange) e are evident during week five of gestation, (II) in week six, distinct tubercles or hillocks (the “hillocks of His”) appear, (III)
the hillocks coalesce, (IV) free ear flap appears caudal to the arch 2 hillock region, (V) during the ninth week, the arch 1 and arch 2 tissue completely merge to obliterate the
branchial cleft; the EAM forms via invagination within branchial arch 1, (VI) the final ear form becomes apparent after the 13th week. The first branchial cleft is marked in II
and III with a white asterisk. The EAM is marked in IVeVI by a white arrowhead. (b) Early mouse embryos (embryonic day 11.5e12.5 [E11.5eE12.5]) showing the early hillocks
and their initial growth and merging. (c) Representative photographs of the auricle from human embryos at approximately 57, 94 and 118 days of gestation (top row) are
compared to roughly equivalently staged auricles from mouse embryos at E13.5, E16, and E18.5. Note that in the mouse the main body of the auricle, which derives from
branchial arch 2, folds over to cover the EAM until after birth. Human conceptal specimens courtesy of the Birth Defects Research Laboratory, University of Washington.
T.C. Cox et al. / European Journal of Medical Genetics xxx (2014) 1e8 3
Please cite this article in press as: Cox TC, et al., The genetics of auricular development and malformation: New findings in model systems
driving future directions for microtia research, European Journal of Medical Genetics (2014), http://dx.doi.org/10.1016/j.ejmg.2014.05.003
malleus and incus bones of the middle ear, the latter two already
known to derive from branchial arch 1 mesenchyme. These ob-
servations suggested that the EAMis instead derived froma distinct
invagination within branchial arch 1 tissue, which was subse-
quently confirmed by fate mapping studies as well as the observed
loss of the EAM in transgenic mice with the duplicated auricular
structures [Minoux et al., 2013].
While the findings of Minoux et al. [2013] provided definitive
data on the origin of the EAM, their interpretation that the
mouse pinna is entirely derived from branchial arch 2 is
misleading because of their imprecise use of the term ‘pinna’. By
definition, the pinna encompasses all external structures of the
ear, including the tragus and root. Patients with mutations in the
coding region of HOXA2 exhibit microtia that spares structures
such as the tragus, presumed derivatives of branchial arch 1
[Alasti et al., 2008; Brown et al., 2013]. We believe structures
orthologous to the tragus are indeed present in the adult mouse
ear (see Fig. 3), although these are somewhat challenging to
recognize in the prenatal period. Upon review of Minoux et al.’s
histological sections through the pinnae of wildtype embryos and
the ‘duplicated ears’ of Hoxa2 mutant embryos, the characteristic
arch 1 hillocks (seen clearly in horizontal sections of control
embryos in Fig. 8E, I, M from Minoux and colleagues' article.) are
no longer evident in the mutant. In the mutant mouse these have
instead undergone a homeotic transformation to arch 2 auricular
structures. Hence their studies demonstrate that Hoxa2-defined
branchial arch 2 derivatives form the “bulk” of the auricular
tissue, as would be predicted by our current model for human
auricular development (Fig. 1a). Further morphological
investigation and fate mapping studies of branchial arch 1
derivatives in late stage mouse embryos or at postnatal stages (if
these mice survive past birth) may be needed to definitively
demonstrate that orthologous arch 1 derivatives such as the
tragus exist in the mouse. Certainly, consistent with our view,
patients with similar mirror-image ‘auricular’ duplications do not
show evidence of a tragus (see Fig. 2c).
4. Clues to the molecular genetics of pre-auricular tags?
For each syndrome associated with microtia where the
responsible gene has been identified (Table 1), a very characteristic,
albeit often highly variable, auricular phenotype is seen (see Fig. 4).
Notably, only a few of these syndromes are associated with pre-
auricular tags; most only exhibit abnormal auricular morphology.
For example, pre-auricular tags are commonly reported inTownese
Brocks syndrome (SALL1) and Branchio-oto-renal syndrome (EYA1,
SIX1, SIX5) but not in CHARGE (CHD7) or TreachereCollins (TCOF1,
POL1RC, POL1RD) syndromes. Can these specific associations tell us
more about the molecular genetic basis of pre-auricular tags in
other conditions, or in purportedly isolated cases of microtia?
Below, we present the case for pre-auricular tags arising as a result
of ectopic expression of a gene or genes that function early in the
genetic program normally specifying the second branchial arch.
Our case originates from the observation that mice ectopically
expressing Hoxa2, in addition to the obvious mirror-image pinna
duplication, also display small ectopic appendages that resemble
pre-auricular tags (Fig. 2b and c; Minoux et al., 2013). Minoux et al.
reported that these small appendages expressed numerous genes
known to be downstream of Hoxa2, including Bmp4, Bmp5, and
Twsg1. None of these genes are specifiers of branchial arch 2
identity but rather are required for proliferation and
differentiation of auricular cartilage and other cell types.
Consistent with this, each of these genes when disrupted in mice
gives rise to simple microtia phenotypes [DiLeone et al., 1998;
Minoux et al., 2013; Petryk et al., 2004] like those seen in most
syndromic forms of microtia (such as LAMM, Miller, Treachere
Collins, CHARGE, and BOF syndromes; Fig. 4). From these
observations, Minoux et al. concluded that these appendages
were additional incompletely developed auricular structures. In
the clinical setting, the majority of pre-auricular tags, or acces-
sory auricular anomalies as they are sometimes called [Yang et al.,
Fig. 3. Schematic representation of adult (a) human and (b) mouse auricles. The labeling of structures of the mouse pinna was adapted from Theiler and Sweet [1986]. The obscured
opening to the mouse EAM is enlarged in the red rectangle and the tragus represented transparently to better show the EAM location.
Table 1
Major microtia syndromes for which the causative gene(s) has been identified.
Syndrome Gene(s) identified
Auriculo-condylar PLCB4, GNAI3
Branchio-oculo-facial (BOF) TFAP2A
Branchio-oto-renal/Branchiootic (BOR/BO) EYA1, SIX1, SIX5
CHARGE CHD7, (SEMA3E)
Fraser FRAS1, FREM2, GRIP1
Kabuki MLL2, KDM6A
KlippeleFeil GDF6
Labyrinthine aplasia, microtia and
microdontia (LAMM)
FGF3
Lacrimo-auriculo-dento-digital (LADD) FGFR2, FGFR3, FGF10
Mandibulofacial dysostosis with
microcephaly
EFTUD2
MeiereGorlin (Ear-patella-short stature) ORC1, ORC4, ORC6, CDT1, CDC6
Microtia, hearing impairment, and
cleft palate
HOXA2
Miller DHODH
Nager SF3B4
Oculo-auricular (OA) HMX1
TowneseBrocks SALL1
TreachereCollins TCOF1, POL1RC, POL1RD
T.C. Cox et al. / European Journal of Medical Genetics xxx (2014) 1e8 4
Please cite this article in press as: Cox TC, et al., The genetics of auricular development and malformation: New findings in model systems
driving future directions for microtia research, European Journal of Medical Genetics (2014), http://dx.doi.org/10.1016/j.ejmg.2014.05.003
2006], contain cartilage [Brownstein et al., 1971] that would
support their origination from ectopic activation of the normal
auricular developmental program.
We posit that similar ectopic activation of the HOXA2 (branchial
arch 2) genetic program may explain the presence of pre-auricular
tags whether in the aforementioned syndromes or in sporadic
cases. For example, SALL1, which encodes one of the four human
homologs of the Drosophila region-specific homeotic gene spalt
[Jürgens, 1988; De Celis and Barrio, 2009], is generally believed to
function as a global transcriptional repressor. Spalt was first
identified because its mutation resulted in partial homeotic
transformation of both the head and tail end of Drosophila. In
invertebrates, spalt genes have been found to be direct targets of
members of the archetypal Homeobox (Hox) factors but also are
themselves involved in regulation of expression of various
homeobox genes, including both archetypal and orphan homeobox
genes [Copf et al., 2006; Toker et al., 2003]. This complex
regulatory network is required to specify the identity of different
segments of the invertebrate body plan. In at least some
mammalian cell types, a similar complex relationship with
homeobox genes is apparent for SALL1/Sall1. In the mouse limb
bud, the expression domain of Sall1 is regulated by the concerted
activities of Hoxa13 and Hoxd13 [Kawakami et al., 2009], while in
embryonic stem cells Sall1 appears to repress various Hox genes,
including Hoxd13 and Gsc (Goosecoid) [Karantzali et al., 2011].
Notably, Gsc is expressed in the first and second branchial arches
and mice null for Gsc show small pinnae and absence of the EAM
[Yamada et al., 1995]. Although it is not known whether Sall1 has a
similar regulatory relationship with Hoxa2 in the branchial arches
as it does with Hox proteins elsewhere in the body, it is expressed
early in head mesenchyme prior to the appearance of the branchial
arches and then becomes restricted around the first branchial cleft
[Buck et al., 2001] in the vicinity of the boundary of Hoxa2 at the
time when the hillocks appear. Furthermore, the finding that Hoxa2
contributes to and maintains Gsc expression in branchial arch 2 but
not arch 1 mesenchyme [Grammatopoulos et al., 2000] is also
consistent with a regulatory relationship establishing arch identity.
Another intriguing relationship is that found between SALL1 and
the proteins mutated in Branchio-oto-renal syndrome: SIX1, SIX5
and EYA1. Sall1/SALL1 in different developmental contexts is known
to be transcriptionally-activated by Six1/SIX1 [Chai et al., 2006; Xu
et al., 2003]. Six1 and Eya1, and likely Six5 and Eya1, interact and
function as a transcriptional complex [Ahmed et al., 2012; Guo
et al., 2011] that likely directly regulates Sall1. Thus, each of the
genes linked to microtia syndromes in which pre-auricular tags
are a prominent feature may interact to limit expression of the
HOXA2-directed genetic program to the second branchial arch.
If such relationships are indeed present in the branchial arches,
it is not difficult to imagine that mutations in these key, upper-level
transcriptional regulators may perturb the underlying genetic
programs set up by homeobox genes that define branchial arch 1
and 2. Consistent with this notion, as seen in Fig. 4a, some Townese
Brocks syndrome patients positive for a SALL1 mutation can also
present with partial mirror-image auricular duplications. Like-
wise, loss-of-function mutations in SIX1/5 (or its cofactor, EYA1)
that cause BOR syndrome, could be expected to lead to reduced
SALL1 expression, explaining the reported ectopic auricular ap-
pendages. Such mutations may not only disrupt the tight regulation
of the boundary of expression of the Hoxa2 program, but also could
feasibly reduce overall expression of the regulatory network
throughout the arches. Even subtle reduction in expression within
branchial arch 2 mesenchyme may be sufficient to result in dys-
morphology of arch 2-derived auricular structures and could
explain the co-existence of microtia phenotypes and pre-auricular
tags, and in turn supporting the predictive value of pre-auricular
tags in determining the familial risk of microtia [Campana et al.,
2013; Luquetti et al., 2012].
Of interest, pre-auricular tags are also a common feature of the
OAV spectrum as well as Oculo-auriculo-fronto-nasal syndrome
(OAFNS) for which the causative gene(s) have not yet been iden-
tified [Evans et al., 2013; Heike and Hing, 2009]. It will therefore be
of much interest to see whether genes for these disorders also
encode important upper-level regulators that help specify first
and second branchial arch identities.
Fig. 4. Auricular dysmorphism in syndromic microtia patients with confirmed genetic diagnoses. a. Comparison of ear presentations from patients with different types of syndromic
microtia. In most cases the external ear morphology is very specific, and the images representative, for each syndrome. b. In some disorders, such as TreachereCollins syndrome, the
auricular malformation can vary greatly. In some cases the ears in different patients are strikingly similar (top row) while others bear little similarity (bottom row). Photographs of
patient ears for each of the following syndromes: CHARGE, Auriculo-condylar, Miller, BOF, and TreachereCollins syndrome, were generously provided by Prof Michael Cunningham
(Seattle Children’s Craniofacial Center). The image of auricular phenotype in LAMM syndrome was reproduced from GeneReviews (www.ncbi.nlm.nih.gov/books/NBK1116/;
copyrighted to University of Washington, Seattle).
T.C. Cox et al. / European Journal of Medical Genetics xxx (2014) 1e8 5
Please cite this article in press as: Cox TC, et al., The genetics of auricular development and malformation: New findings in model systems
driving future directions for microtia research, European Journal of Medical Genetics (2014), http://dx.doi.org/10.1016/j.ejmg.2014.05.003
5. Are EAM anomalies also products of disrupted branchial
arch identity?
Suspected partial EAM duplications, akin to that seen in the
mice ectopically expressing Hoxa2, have been described in patients
with congenital aural atresia, pre-auricular skin tags, and small or
dysmorphic auricles [Blevins et al., 2003]. Because the first cleft had
long been thought to be the only branchial cleft to persist and
produce an adult structure, such anomalies were reasonably
attributed to ectopic branching during formation of the EAM
from the first branchial cleft [Blevins et al., 2003]. The current
classification schemes that are based on this assumption should
now be re-evaluated in light of the data reported by Minoux et al.
[2013]. In our view, such presentations could be explained by
reduced expression levels or activity of HOXA2 or its immediate
downstream effectors in arch 2. A precedent for such altered
downstream effectors leading to complete or partial branchial
arch homeotic transformation can be found in Auriculo-condylar
Syndrome (ACS). The craniofacial phenotype of ACS patients is
considered to be a result of homeotic transformation of the
mandible to a maxilla, with additional characteristic auricular
anomalies (a “question-mark” ear). Rieder et al. [2012], employing
exome sequencing on a collection of carefully phenotyped patients,
recently identified mutations in two separate genes, PLCB4 and
GNAI3, that encode core signaling molecules of the EDN1-DLX5/6
pathway, as the basis of ACS.
6. Guiding genetic studies in isolated microtia
Under our model, mirror-image auricular duplications and pre-
auricular tags in patients would result from ectopic expression of
Hoxa2 or its immediate downstreameffectors within branchial arch
1, whereas complete or partial EAM duplications would arise from
decreased or loss of Hoxa2 or its immediate downstream effectors
in arch 2. We have presented an argument for how this may occur
through mutations in SALL1, SIX1, SIX5 and EYA1. However, coding
region mutations affect the protein in all cells and tissues in which
it is expressed, hence the broader array of phenotypes seen in these
syndromes. So, how might these auricular anomalies occur in
isolation? Given the highly variable presentation seen in these
syndromes, it is possible that coding region mutations in the same
genes may contribute to the incidence of isolated auricular dupli-
cations and pre-auricular tags with or without associated microtia.
It is also feasible that somatic mutations in the genes contribute to
the incidence, as has recently been shown for other disorders
[Guerrini, 2012; Mirzaa et al., 2013]. However, decreased
expression of genes such as SIX1 or EYA1, or even ectopic
expression of HOXA2, as a consequence of mutation of non-
coding regulatory regions could also produce the same result. Ev-
idence in support of such a mechanism in microtia-related phe-
notypes, as explained below, has recently come from somewhat
unexpected sources.
Hmx1 is a homeodomain transcription factor that was first
identified in the developing mouse and chick nervous system and
eye [Wang and Lufkin, 2005], where it plays a critical role in the
development of peripheral autonomic and sensory neurons
[Furlan et al., 2013; Quina et al., 2012a]. In the mouse, Hmx1
expression appears in the branchial arches at approximately
E10.5, well after the positional Hox code in this region of the
embryo is established [Yoshiura et al., 1998]. Despite the initial
identification of Hmx1 in the nervous system, forward genetic
studies in mice identified mutations in Hmx1 in both the
“dumbo” (dmbo) and “misplaced ears” (mpe) mutant lines
[Munroe et al., 2009]. The dmbo and mpe mutants are
characterized by ocular defects as well as malformed and
ventrally displaced and rotated pinnae. The unusual ear
morphology and position gave the appearance of protruding ears,
reminiscent of the cartoon elephant that gives the dumbo strain
its name. The recessive mouse dmbo and mpe alleles consist of a
nonsense mutation and an 8 bp coding region deletion,
respectively, and are thus loss-of-function alleles. In humans, a
coding variant of the HMX1 gene underlies a recessive disorder
called oculo-auricular syndrome (OAS), characterized by malfor-
mations of the pinna and variable eye defects [Schorderet et al.,
2008; Vaclavik et al., 2011]. The human HMX1 allele associated
with OAS consists of a 26 bp deletion in the coding region,
resulting in a frameshift and also a likely null allele.
Recently, mutations in the Hmx1 locus have also been identified
in rats and cows, leading to what appears to be isolated ear mal-
formations. However, rather than a mutation of the Hmx1 coding
sequence, the isolated pinna phenotypes in both species have been
attributed to disruption of the same conserved non-coding element
(CNE) downstream from the Hmx1 gene. The recessive rat dumbo
(dmbo) auricular phenotype strongly resembles its mouse coun-
terpart [Kuramoto et al., 2010]. This rat auricular phenotype was
found to be caused by a 5777 bp deletion residing w80 Mb
downstream of the Hmx1 transcription unit [Quina et al., 2012b].
A CNE of w300 bp within this region exhibits very high identity
(85e98%) between all mammalian species, and shares a core of
conserved sequence that is even retained in reptiles, fish and
amphibians. Importantly, in the developing dumbo rat embryo,
Hmx1 protein expression is lost in branchial arch mesenchyme
that contributes to the pinna and its supporting structures, but is
preserved in sensory neurons. This finding strongly suggests that
the Hmx1 distal CNE functions as a tissue-specific enhancer regu-
lating Hmx1 expression in the lateral facial mesenchyme that
contributes to auricular development. The ‘crop ear’ trait that is
common in the Highland cattle breed represents a moderately to
severely truncated (or cropped) ear deformity, which may vary
according to gene dosage and genetic background [Scheider et al.,
1994]. In contrast to the rat dumbo phenotype, the bovine crop
ear trait exhibits partially dominant inheritance and is due to a
76 bp duplication within the most highly conserved part of the
Hmx1 distal CNE [Koch et al., 2013].
While there have been a small number of regulatory region
variants identified at other loci that are presumed to cause the
associated phenotypes, providing proof that such non-coding var-
iants are causative can be challenging. This example of mutations in
the same Hmx1 CNE in different species yielding similar pheno-
types provides some of the strongest evidence to date for non-
coding, regulatory elements playing an important role in more
‘isolated’ disease presentations [Turner and Cox, 2014].
Importantly, it also highlights that CNE mutations, or for that
matter any non-coding mutations, in HMX1 or any other syn-
dromic microtia gene could be sufficient to cause isolated microtia
phenotypes in humans. Such ‘regulatory’ mutations may however
have very different phenotypic outcomes (in terms of penetrance
and range of affected tissues) depending on the regulatory element
involved. For example, regulatory element mutations could elimi-
nate expression of a given gene in selective tissues and spare others
(as was the case with Hmx1). Alternatively, such mutations could
result in increased or ectopically activated expression (i.e. de-
repression of genes) through disruption of binding of a transcrip-
tional repressor. In the case of SIX1/5, EYA1, SALL1 or even HOXA2,
such alterations in expression levels or tissue distribution may be
sufficient to perturb the boundary of expression of the arch 2
program and thus result in pre-auricular tags, EAM duplications or
stenosis, or for larger perturbations, even partial or complete
mirror-image auricular duplications. In such situations, the un-
derlying cause would not be detectable using standard exome
T.C. Cox et al. / European Journal of Medical Genetics xxx (2014) 1e8 6
Please cite this article in press as: Cox TC, et al., The genetics of auricular development and malformation: New findings in model systems
driving future directions for microtia research, European Journal of Medical Genetics (2014), http://dx.doi.org/10.1016/j.ejmg.2014.05.003
sequencing approaches. Thus, the development of alternative
sequencing strategies, for example, targeted sequencing of a
“CNEome” or a means to bioinformatically assess CNE’s within
whole genome sequencing data, and/or high-resolution copy
number variant detection strategies, may ultimately be more
lucrative for screening cases with ‘isolated’ or non-syndromic
phenotypes.
7. Concluding remarks
The advances in, and decreasing costs of, genome sequencing
technologies are offering astonishing opportunities to identify the
causes of human disorders, and have already proven a boon for
studies of syndromic forms of microtia. However, current
sequencing approaches have not yet proven successful in ‘isolated’
microtia, although recent reports of some large multigenerational
families with many affected individuals [Zhang et al., 2010] may
increase chances of identifying the causative genes. In addition,
as many as 1 in 10 OAVS cases are reported to be familial [Heike
and Hing, 2009] and again numerous multigenerational families
are being collected at multiple centers raising the prospect of
further insight being available in the near future. However, the
variable penetrance and expressivity in these conditions suggests
confounding non-genetic factors that may prove challenging to
resolve. Prenatal exposure to teratogens such as thalidomide,
mycophenolate mofetil or retinoic acid are already well docu-
mented as causing microtia. However, these are unlikely to explain
the majority of instances of isolated microtia or OAVS. Thus, roles
for other non-genetic or genetic factors are still favored as the main
contributors to susceptibility and indeed phenotypic variability in
the condition. Determining these genetic and epigenetic contri-
butions to isolated auricular malformations is arguably the most
significant challenge for the future.
One key aspect in moving the field forward is the pressing need
in both humans and mouse models for precise and thorough phe-
notyping that includes sufficient descriptive detail, photographic
documentation and quantitative measurements where possible.
Better characterization of ear malformations will facilitate assign-
ment of sub-phenotypes that in turn will likely improve the rate of
gene discovery in microtia. To this end, the availability of new
phenotypic tools where each part of the external ear can be an-
notated and scored should aid and standardize this process
[Luquetti et al., 2013a,b]. The need for detailed phenotyping is
further emphasized with the recent significant amendment of the
model of mammalian outer ear morphogenesis.
The mouse offers many significant advantages for studies of
microtia, including the existence of numerous mutant models
displaying a variety of auricular malformations and the accessibility
of appropriately aged embryos for developmental genetic studies of
external ear development. Ultimately, the publication of new ge-
netic data fromsome surprising and non-traditional animal models
has provided the first support for the role of regulatory element
mutations as a cause of microtia-related phenotypes. These find-
ings prompt a cautious re-evaluation of the utility of ‘default’
exome sequencing strategies in genetic studies of isolated microtia,
and offer some guidance to approaches that might be taken to
further understand the genetics of isolated microtia.
Acknowledgments
We would like to thank Matt LaCourse for technical assistance,
the Birth Defects Research Laboratory at the University of Wash-
ington for human conceptal specimens, and Prof Michael Cun-
ningham (Seattle Children’s Craniofacial Center) for numerous
clinical photos shown in Fig. 4. This work is supported in part by the
Laurel Foundation Endowment for Craniofacial Research (TCC),
grant R01 DE022561 (TCC), grants R01 NS064993 and R01
HD033442 (EET), and R00 DC011282 (DVL). SV is supported by a
Post-Doctoral Fellowship Award from the American Association of
Orthodontics Foundation and a University of Washington, School of
Dentistry Institutional Trainee Award (T90 DE021984).
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