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. Author manuscript; available in PMC: 2015 Aug 1.
Published in final edited form as: Eur J Med Genet. 2014 May 29;57(8):394–401. doi: 10.1016/j.ejmg.2014.05.003

The genetics of auricular development and malformation: new findings in model systems driving future directions for microtia research

Timothy C Cox 1,3,4,6,ˆ, Esra D Camci 1,4, Siddharth Vora 1,4, Daniela V Luquetti 1,3, Eric E Turner 2,5
PMCID: PMC4143470  NIHMSID: NIHMS605936  PMID: 24880027

Abstract

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 developmental 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.

Keywords: microtia, anotia, auricular development, craniofacial microsomia, OAVS, Goldenhar syndrome, mouse models

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 extremes of auricular phenotypes (Hunter et al., 2009). Ectopic structures, known as pre-auricular tags, are also sometimes associated 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 (Nelson and Berry, 1984; Castilla and Orioli, 1986; Mastroiacovo et al., 1995; Shaw et al., 2004; Forrester and Merz, 2005; Suutarla et al., 2007; Canfield et al., 2009; Harris et al., 1996; Gonzalez-Andrade et al., 2010). 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; Shaw et al., 2004; Suutarla et al., 2007; Luquetti et al, 2012). 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 (20-60% depending on the study) have 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 (Mastroiacovo et al., 1995; Harris et al., 1996; Shaw et al., 2004; Canfield et al., 2009; Luquetti et al, 2012). In all instances, this percentage rises further when anomalies considered ‘minor’ are included (Luquetti et al, 2013a).

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-auriculovertebral (OAV) spectrum (OAVS; alternately known as craniofacial microsomia or Goldenhar syndrome) (Heike & 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 Townes-Brocks (Kölhase, 2012) and lacrimo-auriculo-dento-digital (LADD; Ordonez & Tekin, 2012) syndromes and also occasionally seen in branchio-oculo-facial syndrome (BOFS) (Lin & 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.

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 embryos and interpretation of malformations seen in the clinic and in animal models. In the early 1880's, His described six protuberances, now known as the ‘hillocks of His’, in the branchial arches of human embryos (His, 1882). These hillocks or tubercles - three in the first arch and three in the second arch - 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 (Figure 1a). Over the ensuing two months, the auricle assumes its recognizable ‘adult-like’ form (see Figure 1a,c).

Figure 1. Development of auricular form in humans and mice.

Figure 1

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(a) updated model of auricular morphogenesis adapted from Porter & 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 - the mandibular process of branchial arch 1 (dark pink) and arch 2 (orange) – 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 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 IV, V and VI by a white arrowhead. (b) Early mouse embryos (embryonic day 10-11 [E10-E11]) showing the early hillocks and their initial growth and merging. (c) Representative photographs of the auricle from human embryos at approximately 57 days, 94 days 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.

From the assessment of various embryonic stages, His concluded that each of the early hillocks contributes to a discrete component of the pinna through precisely timed growth and differentiation (His, 1882; 1885). 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, Muldenhauer (1877) reported similar hillocks, although fewer in number, in the chick which lacks pinnae. Schwalbe, in 1891, also described the presence of branchial tubercles in reptiles that also do not possess auricles (Schwalbe, 1891). These and other studies were nicely summarized in a comprehensive review by George Streeter in 1922, who himself began to question His' model of auricular development (Streeter, 1922). 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; Figure 1a) (Porter & Tan, 2005). Furthermore, as summarized below, new data in mice indicate that the first branchial cleft does not give rise to the EAM.

Auricular morphogenesis: branchial arch specific genetic programs

The revised model of auricular morphogenesis (Figure 1a) also accommodates exciting new molecular genetic findings in mice reported by Minoux and colleagues (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 (Santagati et al, 2005; Minoux et al, 2013). 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 and colleagues subsequently described a family with dominantly inherited non-syndromic bilateral microtia in which they identified a nonsense mutation in HOXA2 (Brown et al, 2013). The auricular features of both families were similar, however affected individuals in the Iranian family presented 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 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 and colleagues 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 (Figure 2a-c), 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.

Figure 2. Mirror-image auricular duplications: a role for ectopic expression of the HOXA2 genetic program?

Figure 2

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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 a, b and c (albeit flipped in orientation from their original presentation) are reproduced from Minoux et al (2013) with permission from the journal Development. doi: 10.1242/dev.098046.

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 investigators found that in addition to the absence of auricles, Hoxa2-deficient mice exhibited duplication of the EAM as well as the malleus and incus bones of the middle ear, the latter two already known to derive from branchial arch 1 mesenchyme. These observations suggested that the EAM is instead derived from a distinct invagination within branchial arch 1 tissue, which was subsequently 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 and colleagues (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 Figure 3), although these are somewhat challenging to recognize in the prenatal period. Upon review of Minoux and colleagues' 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 their Fig 8E,I,M) 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 (Figure 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 Figure 2c).

Figure 3. Schematic representation of adult (a) human and (b) mouse auricles.

Figure 3

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The labeling of structures of the mouse pinna was adapted from Theiler & 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.

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 Figure 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 in Townes-Brocks syndrome (SALL1) and Branchio-oto-renal syndrome (EYA1, SIX1, SIX5) but not in CHARGE (CHD7) or Treacher-Collins (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.

Table 1.

Syndrome Gene(s) identified
Auriculo-condylar PLCB4, GNAI3
Branchiooculofacial (BOF) TFAP2A
Branchiootorenal / Branchiootic (BOR / BO) EYA1, SIX1, SIX5
CHARGE CHD7, (SEMA3E)
Fraser FRAS1, FREM2, GRIP1
Kabuki MLL2
Klippel–Feil GDF6
Labyrinthine aplasia, microtia and microdontia (LAMM) FGF3
Lacrimo-auriculo-dento-digital (LADD) FGFR2, FGFR3, FGF10
Mandibulofacial dysostosis with microcephaly EFTUD2
Meier–Gorlin (Ear-patella-short stature) ORC1, ORC4, ORC6, CDT1, CDC6
Microtia, hearing impairment, and cleft palate HOXA 2
Miller DHODH
Nager SF3B4
Oculo-auricular (OA) HMX 1
Townes–Brocks SALL1
Treacher Collins TCOF1, POL1RC, POL1RD
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Figure 4. Auricular dysmorphism in syndromic microtia patients with confirmed genetic diagnoses.

Figure 4

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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 Treacher-Collins 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, Auriculocondylar, Miller, BOF, and Treacher-Collins 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).

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 (Figure 2b,c; Minoux et al, 2013). Minoux and colleagues reported that these small appendages expressed numerous genes known to be downstream of Hoxa2, including Bmp4, Bmp5, and Tsg. 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 (Minoux et al, 2013; DiLeone et al, 1998; Petryk et al, 2004) like those seen in most syndromic forms of microtia (such as LAMM, Miller, Treacher-Collins, CHARGE, and BOF syndromes; Figure 4). From these observations, Minoux and colleagues concluded that these appendages were additional incompletely developed auricular structures. In the clinical setting, the majority of pre-auricular tags, or accessory auricular anomalies as they are sometimes called (Yang et al, 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 & 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 (Toker et al, 2003; Copf et al, 2006). 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 (Xu et al, 2003; Chai et al, 2006). Six1 and Eya1, and likely Six5 and Eya1, interact and function as a transcriptional complex (Guo et al, 2011; Ahmed et al, 2012) 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 Figure 4a, some Townes-Brocks syndrome patients positive for a SALL1 mutation can also present with partial mirror-image auricular duplications. Likewise, 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 appendages. Such mutations may not only disrupt the tight regulation of the boundary of expression of the Hoxa2 program, but could also 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 dysmorphology 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 (Luquetti et al, 2012; Campana et al, 2013).

Of interest, pre-auricular tags are also a common feature of the OAV spectrum as well as Oculo-auriculofronto-nasal syndrome (OAFNS) for which the causative gene(s) have not yet been identified (Heike & Hing, 2009; Evans et al, 2013). 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.

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 and colleagues (2013). In our view, such presentations could be explained by reduced expression level 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 Auriculocondylar 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 and colleagues, 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 (Rieder et al, 2012).

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 downstream effectors 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 duplications 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 et al, 2004; 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. Evidence in support of such a mechanism in microtia-related phenotypes, 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 (Figure 5f), 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 8bp 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 oculoauricular syndrome (OAS), characterized by malformations 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 26bp 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 malformations. 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 noncoding element (CNE) downstream from the Hmx1 gene. The recessive rat dumbo (dmbo) auricular phenotype strongly resembles its mouse counterpart (Kuramoto et al., 2010). This rat auricular phenotype was found to be caused by a 5,777bp deletion residing ∼80Mb downstream of the Hmx1 transcription unit (Quina et al., 2012b). A CNE of ∼300bp within this region exhibits very high identity (85-98%) 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 regulating 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 76bp 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 variants are causative can be challenging. This example of mutations in the same Hmx1 CNE in different species yielding similar phenotypes 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, 2013). Importantly, it also highlights that CNE mutations, or for that matter any non-coding mutations, in HMX1 or any other syndromic 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 eliminate 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 (ie. de-repression of genes) through disruption of binding of a transcriptional 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 underlying cause would not be detectable using standard exome 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.

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 & 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 documented 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 contributions 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 phenotyping that includes sufficient descriptive detail, photographic documentation and quantitative measurements where possible. Better characterization of ear malformations will facilitate assignment 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 annotated and scored should aid and standardize this process (Luquetti et al., 2013c). 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 genetic data from some surprising and non-traditional animal models have provided the first support for the role of regulatory element mutations as a cause of microtia-related phenotypes. These findings prompt a cautious reevaluation 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 Washington for human conceptal specimens, and Prof Michael Cunningham (Seattle Children's Craniofacial Center) for numerous clinical photos shown in Figure 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 Organization of Orthodontics Foundation and a University of Washington, School of Dentistry Institutional Trainee Award (T90 DE021984).

Footnotes

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