The role of genetic factors in microtia: A systematic review

Introduction

Microtia is a congenital malformation of the outer ears caused by improper embryonic development.^1,2 It is distinguished by small, irregularly shaped external ears, and it can occur unilaterally or bilaterally.¹ The prevalence of microtia varies among ethnic groups (0.83–17.4 per 10,000 births).^2–4 Microtia occurs unilaterally in 80%–90% of cases and bilaterally in 10%–20% of cases.^2,4 Boys are approximately twofold more likely than girls to have microtia, and the right–left bilateral ratio is typically 6:3:1.^2,3 Roughly one-third to one-half of patients with microtia have craniofacial microsomia.^2,3 Microtia is easily misdiagnosed during pregnancy.^5,6 If pregnancy ultrasonography suggests microtia, the diagnosis is easily confirmed and diagnosed following birth based on physical examination.^5,6

The etiology of microtia and the causes of its variations remain unknown.⁴ Although there is compelling evidence that environmental factors such as maternal sociodemographic variables, multiple gestation, diseases (gestational diabetes, cold-like syndrome), and related drug treatments such as isotretinoin use during pregnancy play roles, genetic factors are also believed to influence the embryonic development of microtia.^7–9 Estimates of the prevalence of inherited microtia vary greatly, ranging from 3 to 34%.¹⁰ Although certain studies discovered candidate genetic variations for microtia, no causal genetic mutation has been identified.¹¹

Research on animals with isolated microtia as a prominent feature revealed mutations in homeobox A2 (HOXA2), sine oculis homeobox (SIX), eyes absent transcriptional coactivator and phosphatase (EYA), TBX1, IRF6, and CHUK.¹² Genes identified to be involved in the development of major syndromic microtia were PLCB4 and GNAI3 for auriculo-condylar syndrome; TFAP2A for branchio-oculo-facial (BOF) syndrome; SIX1 and SIX5 for branchio-oto-renal (BOR) syndrome; CHD7 (SEMA3E) for CHARGE syndrome; FRAS1, FREM2, and GRIP1 for Fraser syndrome; MLL2 and KDM6A for Kabuki syndrome; GDF6 for Klippel–Feil syndrome; fibroblast growth factor 3 (FGF3) for labyrinthine aplasia, microtia and microdontia (LAMM) syndrome; FGFR2, FGFR3, and FGF10 for lacrimo-auriculo-dento-digital syndrome; EFTUD2 for mandibulofacial dysostosis with microcephaly; ORC1, ORC4, ORC6, CDT1, and CDC6 for Meier–Gorlin syndrome; HOXA2 for microtia, hearing impairment, and cleft palate; DHODH for Miller syndrome; SF3B4 for Nager syndrome; H6 family homeobox 1 transcription factor gene (HMX1) for oculo-auricular syndrome (OVAS); SALL1 for Townes–Brocks syndrome; and Treacher–Collins–Franceschetti syndrome 1 (TCOF1), POL1RC, and POL1RDT for Treacher–Collins syndrome (TCS).²

FGF3 mutations are commonly found in LAMM syndrome.^13,14 The FGF3 protein regulates a cascade of chemical processes inside cells by binding to its receptor, thereby signaling cells to undergo particular changes, such as proliferating or maturing to perform specialized activities.¹⁵ TCOF1 mutations can cause TCS in up to 78% of patients.¹⁶ HOXA2 encodes key developmental transcription factors of the second branchial arch, which contributes significantly to the development of the external and middle ear in embryonic development, and it was previously linked to autosomal recessive bilateral microtia.^10,17

Because of the unclear role of genetic factors in microtia, we conducted the first systematic review to qualitatively identify the most important genes in the development of microtia. This may help to improve our understanding of microtia, underline the necessity of gene screening and even make prevention of microtia in the near future possible with the help of gene modification.

Methods

Results

All supplementary files can be found in the Extended data.⁷²

Systematic review outline

We discovered 3,742 articles after searching six electronic bibliographic databases. In total, 183 publications were removed because they were not available in English, they were animal studies, their full text was not available, or they were duplicated studies. Only 55 articles were eligible for more extensive evaluation after title and abstract screening. Only 40 papers were included in this study after a comprehensive full text analysis (Figure 1). The included studies were reviewed, utilizing a checklist questions form provided by JBI based on the studies’ methodology. Based on the JBI Tools for case reports, case series, and case controls, all publications involved were assessed as low-risk bias (Supplementary Tables 2–4).

Figure 1. Systematic review flow diagram of included studies.

Discussion

Microtia is a congenital external ear deformity that can range in severity from minor anatomical problems to full ear absence (anotia). Microtia can be a single birth abnormality or part of a broader set of defects or syndrome.⁸ This systematic review attempted to describe the genes that play important roles in the development of syndromic and non-syndromic microtia. Only 40 studies on genetically linked microtia met our selection criteria. In this study, China had the highest number of microtia cases. The findings of this study contradict epidemiological data reported by Klokars et al., who recorded the highest prevalence of microtia in Quito, Ecuador, South America, (17.4/10,000),³ and Luquetti et al., who stated that the highest prevalence of microtia was in Finland, North Europe (4.3/10,000).² This could be because China has a higher overall birth rate than other countries²; therefore, even if it has a greater number of microtia cases, the prevalence rate is low.

We discovered that most patients with microtia had a family history of the disease, including 56.4% of patients with isolated microtia and 80.8% of patients with syndromic microtia. This is consistent with the existing literature, which describes Mendelian hereditary variants of microtia with an autosomal dominant or recessive mode of inheritance.^1,4,20 The rates of familial microtia ranged from 3 to 34%.¹⁰

Based on the gender classification of each study, we discovered nearly 60% of all patients were male, including 59% of patients with syndromic microtia and 58.4% of patients with isolated microtia. In prior research, microtia was more common in male patients than in female patients, with a sex ratio of 1.5:1 and an estimated 20–40% greater risk in males than in females.^4,21

Although microtia can arise bilaterally, 77–93% of patients have unilateral involvement.^4,21 The most common form of syndromic microtia is bilateral microtia.¹ Although bilateral microtia was more common in this study, the type of microtia was only known for 29.6% of subjects. Because of the inadequate data, this could represent a biased outcome for the most prevalent type of microtia.

Most published research on microtia reported the existence or absence of microtia and/or anotia with no further details on severity. Marx (1926), Weerda (1988), Roger (1977), Tanzer (1978), and Hunter (2009) all provided classifications of microtia.⁵ Their classification system was nearly identical, consisting of four grading levels.^4,22 The lobule type, grade III microtia, was the most common microtia type in the literature, accounting for 60% of all cases. The most prevalent severity of syndromic microtia was grade I owing to the high rate of mutations in FGF3, which can cause LAMM syndrome with a grade I microtia presentation.^13,14,23,24

From all examined studies involved, the most common genes associated with microtia were HOXA2, FGF3, and TCOF1. We discovered that in syndromic microtia, the most common types of mutation were TCOF1 deletion (46.9%), and missense and deletion in FGF3 (38%), whereas in isolated microtia, the most common type of mutation in HOXA2 was silent mutation (54.2%). This was consistent with the literature, which indicated that the most common genes involved in microtia were HOXA2, FGF3, HOXD, ORC1, ORC4, ORC6, CDT1, CDC6, DHODH, HMX1, EYA1, and TCOF1.²

FGF3 encodes a protein member of the FGF family.¹⁵ The FGF family has extensive mitogenic and cell survival activities and is involved in various biological processes such as embryonic development, morphogenesis, tissue repair, cell growth and inner ear formation in mice and chicken.¹⁵ FGF3 activates a cascade of chemical reactions inside cells that activate certain changes, such as dividing or maturing to take on specialized functions, by attaching to another protein known as a receptor.¹⁵ FGF3 haploinsufficiency could also be related with dental and hearing problems. FGF signaling is required for the appropriate development of the otic placode, a thickening of the ectoderm on the outer surface of a developing embryo from which the ear develops.²⁵ FGF3 mutation is often found in syndromic microtia, mainly in LAMM syndrome. Congenital deafness with LAMM syndrome is an autosomal recessive disorder characterized by significant bilateral congenital sensorineural deafness coupled with inner ear defects, grade I bilateral microtia, and microdontia (small teeth) as its major phenotypic features.^10,26 The finding of biallelic pathogenic mutations in FGF3 on molecular genetic testing confirms the diagnosis of LAMM syndrome in the proband.^13,14,23,24

The TCOF1 gene, which encodes a suspected nucleolar phosphoprotein known as treacle, has been identified as the cause of TCS,^10,27 an autosomal dominant craniofacial development disorder, in up to 78% of patients.^16,28,29 Inhibition of mature RNA ribosomal (rRNA) production and gene transcription in neural folds prefusion during the early stage of embryogenesis may cause abnormal development due to treacle haploinsufficiency, caused by mutation in the TCOF1 gene, thus affecting proliferation and proper differentiation of these embryonic cells.³⁰ To date, more than 50 mutations have been identified in the TCOF1 gene, most of which are insertions or deletions. TCS is characterized by cleft palate, hypoplasia of facial bones (particularly the mandible and zygomatic complex), downward slanting of palpebral fissures with colobomas of lower eyelids, external ear deformity, conductive hearing loss, and defects in brain development such as microcephaly and mental retardation.^31–36 TCOF1 was the most prevalent gene found in this systematic review. This was because TCOF1 is a causal gene for TCS, which features microtia as one of its clinical symptoms.³² In addition to clinical findings, TCS diagnosis is also confirmed by detection of pathogenic variants of TCOF1, POLR1D, or POLR1B using molecular genetic testing, mainly inherited in an autosomal dominant pattern.³⁷ In accordance with our result, TCOF1 gene deletions were reported to range from a single exon to a whole gene. Despite the fact that >97% of reported cases contained a pathogenic mutation identifiable by sequencing, Bowman et al. (2012) reported 5% of cases (5/92) with a big deletion, suggesting that the rate of large deletions may be higher than current data suggest.³⁸

Homeobox genes participate in the formation of the pharyngeal arches.^4,39 They encode transcription factors that determine cell positional identity and morphogenesis during development, as well as switch on cascades of other genes.^4,39–41 The Hox gene family was discovered to be grouped inside the genome and ordered on the chromosome in the order of expression during development. This ordered pattern of gene expression could be part of a mechanism that generates morphogenetic specification.^4,39 HOXA2 was discovered to be highly expressed in the second brachial arches (BA2), to express critical developmental transcription factors BA2, to play an important role in the development of the external and middle ear during embryonic development, and to be associated with autosomal recessive bilateral microtia as a member of the HOX gene family.^10,17,39 In humans, abnormal or lost HOXA2 function, as well as early and late HOXA2 inactivation, results in auditory system malformations, primarily in the external and middle ear, such as a duplicated or absent auricle^10,39 Consequently, HOXA2 has been proposed as a key transcriptional regulator of auricle morphogenesis.³⁹ Individuals with a homozygous HOXA2 mutation have far more severe clinical symptoms than those with a heterozygous mutation. In a mouse model, inactivation of Hox2 early in development results in the absence of the pinna, whereas late inactivation results in a hypomorphic auricle.⁴²

Aside from HOXA2, FGF3, and TCOF1, other genes linked to microtia include HOXD, ORC1, ORC4, ORC6, CDT1, CDC6, DHODH, HMX1, and EYA1.² Among these, HMX1 located on chromosome 4p16.1 is prominent. It is involved in the differentiation of the lateral facial mesenchyme downstream of embryonic patterning genes.^9,43 In humans, recessive loss-of-function mutations in HMX1 have been linked to OVAS, which is characterized by external ear and eye deformity.⁹ In a five-generation Chinese family with isolated bilateral microtia, a 10-Mb linkage locus covering 4p16 was discovered.^9,43,44

We hypothesized that a link existed between the types of genes involved and the grade of microtia. For example, FGF3 deletions in LAMM syndrome have been clinically identified as grade I microtia.^13,14,23,24 According to the MARX classification,¹⁰ the HOXA2 gene was common in the form of microtia type II and was exclusive to isolated microtia.^{1,17,20,39,45–48} However, no specific type of microtia has been linked to the deletion of TCOF1.^10,49

The field of genetic variables in microtia research is still relatively extensive. More research on genetic variables that contribute to microtia is required, particularly using the next generation sequencing (NGS) and DNA microarray approach. NGS, massively parallel sequencing, or deep sequencing are all terms that refer to DNA sequencing technology that has transformed genomic research.^48,49 In comparison to other technologies, NGS can sequence the entire human genome in a single day. Genomes may be examined without prejudice, allowing mosaic mutations to be detected.⁴⁸ On the other hand, DNA microarray is a revolutionary technique for gene expression profiling.⁵⁰ Microarrays were created as a method for mapping and sequencing vast amounts of DNA. DNA microarrays offer a far higher throughput and are less time consuming than previous approaches. By applying NGS and microarray for screening of genetic risk factors in microtia, the diagnosis of microtia could be made earlier and the patients could get a more comprehensive treatment.

Conclusions

According to this study, most cases of microtia (75.5%) occurred in China, 60.7% of subjects had a family history of microtia, 58.5% of cases occurred in males, 74.2% of cases were bilateral, and 41.3% of cases were grade III microtia. From the studies involved, the three most common genes associated with the development of microtia were HOXA2 (10%), FGF3 (8.4%), and TCOF1 (5.4%). The most prevalent syndromes related to microtia were TCS and LAMM syndrome. Deletion mutations in TCOF1 were found in 15 patients (46.9%), deletion and missense mutations were present in FGF3 in 19 patients (38%), and silent HOXA2 mutations were present in 32 patients (54.2%).

Approximately 76.2% of the genetic factors that contribute to microtia remain unknown. More research on genetic variables in microtia is required, particularly the use of NGS, massively parallel sequencing, or deep sequencing. We recommend that investigations on genetically associated microtia be conducted using observational studies, and the features of patients involved should be described more clearly and comprehensively in the future for better systematic reviews or even meta-analysis.

By understanding the three most dominant genes associated with microtia (HOXA2, FGF3, and TCOF1), we could promote the early screening and detection of microtia in the next generation, allowing us to provide better education and genetic counseling to patients with microtia regarding the possibility of microtia development in their children, and we hope that this systematic review will serve as a reference for the establishment of a global database of patients with microtia.

Data availability