Genetics and Epigenetics in the Genesis and Development of Microtia

Abstract

Microtia is a congenital malformation of the external and middle ear associated with varying degrees of severity that range from mild structural abnormalities to the absence of the external ear and auditory canal. Globally, it is the second most common congenital craniofacial malformation and is typically caused by inherited defects, external factors, or the interaction between genes and external factors. Epigenetics notably represents a bridge between genetics and the environment. This review has devoted attention to the current proceedings of the genetics and epigenetics of microtia and related syndromes.

Microtia (OMIM 600674, OMIM 251800) is a congenital malformation with varying degrees of severity ranging from mild structural abnormalities to the absence of the external and middle ear (Fig. 1). With a global incidence of 0.4 to 8.3/10,000,¹ it is the second most common congenital craniofacial malformation after cleft lip and palate.² Its prevalence varies by region and ethnic group. In China, the incidence of microtia is as high as 3.57/10,000, showing a significant upward trend.³ Microtia affects patients’ appearance and hearing function and is also associated with psychological problems.

FIGURE 1.

Classification of microtia. Microtia is classified into I to IV degrees based on the severity of the auricle malformation.

Microtia is caused by the dysplasia of the first branchial arch and groove and the second branchial arch in the embryonic stage.⁴ The outer and middle ear originates from the mesenchyme through interactions between the first and second branchial arch cells and migrating neural crest cells (NCCs). In the sixth week of pregnancy, the outer ear and ossicles develop. In the first 2 months of pregnancy, the external auditory canal (EAC) originates from the first branchial cleft. Six buds of the first and second branchial arches fuse to form the auricle, a process usually completed at a gestational age of 12 weeks. The pinnae moves from the bottom of the neck to the final, proper position at 20 weeks gestation. With the expansion of the first pharyngeal sac, 3 mature ossicles are wrapped around it to form the middle ear cleft. The outer and middle ear structures completely develop by the 30th week of pregnancy.⁵

Microtia can manifest as an isolated defect, that is, nonsyndromic type, or as part of a syndrome.⁴ Nonsyndromic microtia is a polygenic and complicated disease, while most microtia-related syndromes are monogenic. Inherited defects, external factors, or the interaction between genes and external factors are the main causes of microtia. Epidemiological studies have found that environmental factors, including cold-like syndromes during pregnancy, a history of spontaneous abortion, vaginitis, cervicitis, and other infections, as well as exposure to toxic chemicals, such as polycyclic aromatic hydrocarbons, paints, and pesticides, are risk factors for the disease.⁶ Evidence that strongly suggests a genetic cause for microtia includes the higher frequency of cases in monozygotic twins (38.5%) compared with dizygotic twins (4.5%); reporting of familial cases with autosomal recessive or dominant inheritance patterns linked to variable expression and incomplete penetrance; family history of disease in about 3% to 34% of cases; reporting of >18 different microtia-related syndromes, which include those associated with monogenic defects or chromosomal aberrations; and mouse models proving the genetic cause microtia.⁴ On the basis of reported studies on genetic and environmental factors, and as it represents a bridge between genetics and the environment, epigenetics has become a new discipline that provides a novel way to study the etiology of congenital microtia. Understanding the causes of microtia is very important for clinical intervention.

GENETICS OF MICROTIA

Nonsyndromic Microtia

Nonsyndromic microtia is a polygenic disease with a complex etiology and pathogenesis. Compared with the syndromic type, the genes responsible for cases of it are more difficult to find. The limited number of susceptibility genes currently known include the homeobox A2 (HOXA2) and A1 (HOXA1), bone morphogenetic protein 5 (BMP5), goosecoid homeobox (GSC), and T-box transcription factor 1 (TBX1) genes. The fibroblast growth factor (FGF), retinoic acid (RA), wingless/INT, and BMP signaling pathways are also known to be involved in this condition.

Homeobox A2 has long been considered the primary susceptibility gene for the pathogenesis of microtia,^7,8 and its coding protein belongs to the HOX family. The HOX genes have regulatory roles in many steps involved in the migration and differentiation of cranial NCCs and participate in the cascade reactions of many other genes.⁹ Homeobox A2, engaged in branchio-oto-renal syndrome, partially controls auricle morphogenesis by expressing EYA transcriptional coactivator and phosphatase 1 (EYA1) and BMP signaling pathway.⁸ The inactivation of the Hoxa2 gene in mice results in the EAC’s duplication and the auricle’s absence. Upstream of E11.5, the deletion of Hoxa2 leads to the loss of the auricle, and between E12.5 and E13.5, Hoxa2 deficiency results in a hypomorphic auricle.⁸ Various mutations in HOXA2 detected in sporadic cases and autosomal dominant or recessive families with nonsyndromic microtia events have been reported.^10,11 In addition to HOXA2, HOXA1 plays an important role in regulating the development of the external ear during the early stages of embryonic growth. It has therefore been considered an important microtia-associated susceptibility gene for many years.¹² Hoxa1 null murine mutants manifest with dysplasia of the outer, middle, and inner ear.¹³ However, the clinical evidence for HOXA1 mutation causing microtia is still very limited.

The BMP-associated genes are associated with cartilage growth and repair. Several loss-of-function mutations, the first described in 1921¹⁴ and mapped to the Bmp5 gene in 1992, contribute to the short-ear phenotype in mice.¹⁵ Bmp5 was highly expressed in the proliferative zone chondrocytes of the tibia of mice, and the expression increased with the differentiation of chondrocytes, indicating its importance in chondrocyte differentiation and proliferation.¹⁶ Few reports about BMP5 and its association with nonsyndromic microtia have been reported.^17,18 More studies are needed to confirm this link.

Goosecoid homeobox is a homologous domain transcription factor and a downstream target of endothelin.¹⁹ It plays a critical role during gastrulation in early embryonic development and migration of cranial NCCs.²⁰ Mice with a homozygous disruption of Gsc have a variety of developmental defects, including those affecting the malleus, tympanic ring, and EAC.²¹ In a family with nonsyndromic microtia, 20 out of 56 members were found to harbor a mutation in GSC.²²

Tbx1 gene expression in the first branchial arches, as demonstrated through Tbx1 homozygous mutant mice, plays an important role in forming the middle and external ear.²³ The degree of deformity in the skeletal components of mice varies from mild hypoplasia to complete loss.²⁴ These phenotypes result from a failure in developing the posterior branchial arch and the misdirection of NCCs.²⁴ Currently, only one TBX1 mutation (c.928G > A, p.G310S) has been reported to be responsible for severe microtia-atresia.²⁵ More reports concerning TBX1 and the manifestation of nonsyndromic microtia are needed to prove its pathogenicity.

While the pathogenicity of the genes described here remains to be confirmed, other genes may also cause microtia, according to previous studies (Supplemental Table 1, Supplemental Digital Content 1, http://links.lww.com/SCS/F819).

SYNDROMIC MICROTIA

A wide range of other abnormalities has been described in 27% of patients with microtia, including cleft lip and palate, facial asymmetry, renal abnormalities, cardiac defects, microphthalmia, polydactyly, and deformities of the spine.⁴ The most common syndromes associated with microtia are the oculoauriculovertebral spectrum (OAVS), Treacher Collins syndrome (TCS), and branchio-oto-renal spectrum disorder. This paper briefly introduces some of the most common syndromes.

Oculoauriculovertebral Spectrum

Oculoauriculovertebral spectrum (OMIM 164210), predominantly characterized by asymmetric ear malformations, hemifacial microsomia, ocular defects, and vertebral abnormalities, is a disorder involving craniofacial morphogenesis with a reported prevalence of 1/45,000 to 1/5,600.²⁶ The clinical manifestations of OAVS are diverse. Many terms, including craniofacial microsomia (CFM), hemifacial microsomia, first and second pharyngeal arch anomalies, and Goldenhar syndrome, have been used to designate this complex spectrum of abnormalities. All OAVS patients present with microtia. While OAVS is mostly sporadic, genetic transmission has been found through segregation analysis in some cases. The known pathogenic genes are the myelin transcription factor 1 (MYT1), Zyg-11 family member B (ZYG11B), splicing factor 3b subunit 2 (SF3B2), and the newly discovered forkhead box I3 transcription factor (FOXI3).

MYT1, which contributes to neurodevelopment, was identified by Lopez et al²⁷ among a cohort of 169 OAVS patients for the first time in 2016. Transient knockdown of myt1a (homolog of myt1 in zebrafish) caused specific craniofacial cartilage alterations. In addition, overexpression of cellular wild-type myt1 in this model induced downregulation of RA receptor β (RARB).²⁷ In vitro, functional studies of a novel de novo missense mutation found that MYT1 overexpression downregulated all retinoic acid receptor genes (RARA, RARB, and RARG),²⁸ all of which are involved in retinoic acid-mediated transcription. Therefore, MYT1 may mediate the development of OAVS through the RA pathway.

ZYG11B encodes a member of the E3 ubiquitin ligase complex and participates in substrate recognition of proteasome degradation. Tingaud-Sequeira et al²⁹ firstly found a nonsense mutation of ZYG11B in an OAVS patient. In vitro experiments showed that this mutation resulted in a truncated protein and its subcellular localization was changed. Knockout of homologous genes in zebrafish revealed abnormal craniofacial cartilage and eye development. ZYG11B was regulated by RA the same as the OAVS gene MYT1.²⁸ However, the clinical evidence is not strong enough to fully prove the pathogenicity of the gene, which needs to be further confirmed by expanding the sample.

SF3B2, involved in pre-mRNA splicing as a component of the splicing factor SF3B complex, is the most common causative gene to date, haploinsufficiency of which accounts for ~3% of sporadic CFM.³⁰ Timberlake et al³¹ identified 7 CFM kindreds with loss-of-function variants in SF3B2. Targeted knockdown of Sf3b2 in Xenopus embryos altered neural crest development and caused craniofacial defects, suggesting that NCC depletion is a contributing factor to the pathoetiology of CFM.³¹ However, the molecular mechanisms underlying spliceosomal dysfunction in CFM are poorly understood. Recent studies have clarified the role of spliceosome dysfunction in pathogenic exon retention. These highly conserved alternative exons contain a premature stop codon, usually spliced from pre-mRNA. Although poison exon has been found in many genes that play an important role in development and morphogenesis, its exact function is still unclear.³² It will be interesting to determine whether the retention of poison exons that result from splicing abnormalities caused by the variants in SF3B2F contributes to this disease process.

As a member of the FOX transcription factor family, FOXI3 plays a role in embryonic development, particularly in forming several organs and tissues. Recently, it was reported that FOXI3 variants were detected in 4 CFM pedigrees.³³ A study by Mao et al³⁴ strongly supported the involvement of the likely pathogenic FOXI3 variants in CFM based on the knock-in mouse model. Previous studies have reported that Foxi3 ^–/– mice showed abnormalities in the inner ear, the jaw, external, and middle ear,³⁵ and Foxi3-Gata3 and Foxi3-Fgf8 signaling is essential for facial morphogenesis.³⁶ Although it is clear that FOXI3 is a key regulator of craniofacial development, there remains a gap in our knowledge of the mechanisms by which FOXI3 operates in these processes.

Other possible pathogenic genes screened out in patients with OAVS included SALL1, AMIGO2, and OTX2. ^37–39 However, the pathogenesis of these genes remains to be confirmed.

Treacher Collins Syndrome

Treacher Collins syndrome (OMIM 154500) is a congenital craniofacial malformation mainly resulting from autosomal dominant variants and has an incidence of ~1/50,000.⁴⁰ The main characteristics of the disease include hypoplastic facial bones, microtia or even anotia, micrognathia, and other deformities of the middle and outer ears, auditory pits, hearing loss, and cleft palate. Approximately 60% to 80% of TCS patients present with microtia.⁴ Treacle ribosome biogenesis factor 1 (TCOF1), RNA polymerase I and III subunits C (POLR1C) and D (POLR1D), and RNA polymerase I subunit B (POLR1B) are presently the pathogenic genes identified for TCS. According to genetic etiology, TCS was classified into 4 types: TCS1 caused by TCOF1 mutation (OMIM 154500), TCS2 caused by POLR1D mutation (OMIM 613717), TCS3 caused by POLR1C mutation (OMIM 248390), and TCS4 (OMIM 618939) caused by POLR1B mutation.

TCOF1, first identified using positional cloning in 1996, is involved in the differentiation and proliferation of NCCs in the first and second branchial arches.⁴⁰ The treacle protein, encoded by TCOF1, plays an active role in the facial bone structures and other tissue during early embryogenesis. Studies on Tcof1 expression in mouse embryos supported the role this gene plays in the development of the craniofacial complex and additionally proved that treacle is a phosphoprotein potentially involved in nucleolar-cytoplasmic transport.⁴¹ Heterozygous Tcof1 knockout mouse model presented with severe craniofacial dysmorphia.⁴² Many reviews have described the pathogenesis of TCOF1. As the main pathogenic gene of TCS and present in 78% to 93% of all cases, >250 TCOF1 mutations have been described. Mutations have been identified, including indels, splicing, missense, and nonsense mutations. Most of these variants lead to the premature appearance of a termination codon.⁴³

POLR1C and POLR1D encode the RNA polymerase I and III subunits for rRNA transcription, respectively. Dauwerse et al⁴⁴ were the first to identify the involvement of these 2 genes in TCS and subsequently confirmed the genetic heterogeneity of TCS, thereby providing evidence supporting the hypothesis that it is a ribosomopathic condition. Synonymous with the characteristics of humans with TCS, homozygous mutants of polr1c and polr1d in zebrafish not only exhibited achondroplasia and cranial dysplasia but also demonstrated decreased cellular proliferation capacity and increased apoptosis.⁴⁵ Familial TCS cases with an autosomal recessive inheritance model caused by mutations in POLR1C or POLR1D have been reported.⁴⁶

POLR1B, a subunit of RNA polymerase I, is responsible for the transcription of ribosomal RNA (rRNA) genes and the production of rRNA. Sanchez et al⁴⁷ were the first to find three potential pathogenic single-nucleotide variations in POLR1B. Similar to that found in human TCS cases, the knockdown of polr1b in zebrafish induced an abnormal craniofacial phenotype. Pathogenic variants in POLR1B may induce increased p53-dependent apoptosis in a restricted neuroepithelium area, thereby changing the migration of NCCs and leading to cranioskeletal abnormalities.⁴⁷ POLR1B was considered a new causative gene responsible for a novel form of TCS (TCS4).

Branchio-oto-renal Syndrome

Branchio-oto-renal (BOR) syndrome (OMIM 113650) is an autosomal dominant disease characterized by branchiogenic abnormalities, hearing loss, and renal malformations and has an incidence of ~1 per 40,000 live births. About 30% to 60% of BOR patients present with microtia.⁴ EYA1, SIX homeobox 1 (SIX1), and 5 (SIX5) genes are currently considered to be causative for BOR syndrome.

EYA1 encodes for a component of the eyes absent family of proteins. The encoded protein may be an important contributor to the development of the ears, branchial arches, kidneys, and eyes. Using positional cloning, EYA1 was the first causative gene to be identified for BOR syndrome.⁴⁸ Eya1 homozygous-deficient mice manifest without ears and kidneys, while heterozygous-deficient mice show phenotypes similar to BOR syndrome.⁴⁹ EYA1 gene encodes a multifunctional protein, as a protein tyrosine phosphatase and coactivator of transcription, interacts with 6 transcription factors to maintain protein stability. However, the loss of interaction with 6 proteins after the mutation of EYA1 leads to rapid protein degradation and disease.⁵⁰ EYA1 mutation, including nonsense and missense variants, splice abnormalities, and micro-gene or whole-gene deletions, have been identified in 93% of all BOR syndrome patients.⁵¹

SIX1 participates in the control of embryonic development and cell apoptosis and proliferation. Ruf et al⁵² first found 3 different SIX1 mutations in BOR cases and confirmed that mutations affect the interaction between EYA1 and SIX1. The gene notably contributes to the development of some organs, including the inner ear, muscle tissue, and kidneys, with Six1 mutant mice confirming its critical role in this regard.⁵³ Compared with EYA1 variants, the frequency of SIX1 gene mutations in BOR syndrome patients is lower.

The protein encoded by SIX5 is a transcription factor with a homologous domain that regulates organogenesis. Hoskins et al⁵⁴ were the first to find 4 different heterozygous missense mutations in SIX5. Functional analysis of these variants has indicated that 2 mutations affect the EYA1-SIX5 binding capacity and gene transcription activation through the SIX5 or the EYA1-SIX5 complex.⁵⁴ Although heterozygous mutations in SIX5 were thought to be one cause of BOR syndrome, they have rarely been found in these patients. Notably, no developmental defects occurred in Six5 single knockout mice.⁵⁵ Therefore, the role of SIX5 in BOR syndrome remains to be studied further.

Although these 3 syndromes correlate strongly with microtia, other syndromes and pathogenic genes associated with this condition are listed in Supplemental Table 2, Supplemental Digital Content 1, http://links.lww.com/SCS/F819.

EPIGENETICS OF MICROTIA

Epigenetics is a branch of genetics that studies the heredity of gene expression changes without changing the nucleotide sequence of genes. Although cells in each tissue type share the same DNA sequence, epigenetic regulation differs across cell types. This regulation contributes to cellular differentiation by enabling cells to express only the genes necessary to function in their associated tissue. Epigenetic modifications can persist for a cell’s life and survive division, but they can also be influenced and changed by environmental factors.⁵⁶ Collectively, this implies that external influences may lead to long-term changes in gene activity. Epigenetic processes include DNA and histone modification and regulation of noncoding RNA.⁵⁷ However, limited studies have been conducted on the epigenetics of microtia to date, with research on noncoding RNA, such as circular RNA, and histone modifications being absent.

DNA Methylation

DNA methylation is a common mechanism by which the expression of genes in eukaryotic cells is epigenetically regulated and is the main epigenetic modification of genomic DNA. Since DNA methylation is important for the structure and function of embryonic tissue development, abnormal epigenetic mechanisms may lead to microtia. DNA methylation mediates many environmentally induced changes in gene expression. Although supporting evidence is lacking in humans, animal models have indicated that DNA methylation and other epigenetic processes are critical for facial development. Exposure to 5-azaC, an inhibitor of DNA methyltransferase (DNMT) enzyme activity, can cause microcephaly and craniofacial cartilage malformations in Japanese rice fish. The expression of DNMTs is developmentally regulated. Craniofacial defects may therefore result from dysregulation in DNMT expressions.⁵⁷ Under normal conditions in the human genome, 60% of the promoter regions have unmethylated CpG islands. Where abnormal CpG island methylation is present, gene silencing occurs. Abnormally methylated CpG islands of the COL18A1, MYH14, RBMY1A1, ZIC3, and EYA1 genes have been found and may therefore be associated with the pathogenesis of microtia.⁵⁸ Hypomethylation may also cause microtia.⁵⁹

Noncoding RNA Regulation

Noncoding RNA is a form of RNA that is not translated into protein. The noncoding RNA elements involved in gene regulation include micro RNA (miRNA), long noncoding RNA (lncRNA), circular RNA, piwi-interacting RNA, and small interfering RNA. Of these, miRNA and lncRNA are the most widely studied at present.

MiRNAs, which can control the expression of post-transcriptional genes, are a class of noncoding RNAs that are 20 to 22 nucleotides in length. These small RNA molecules have been shown to control cell growth, differentiation, and apoptosis, and many miRNAs regulate intrinsic or extrinsic apoptotic pathways.⁶⁰ In mouse fetuses, 40 miRNAs are differentially expressed in ear tissue. Some of these elements, such as mmu-miR-10a, have been predicted as miRNAs that target genes related to the development of the external ear⁶¹ but have not been further validated in animal models. The expression of miR-203, miR-486-5p, miR-200c, and miR-451 in microtia-associated tissue was found to vary considerably compared with healthy controls^62,63; these results may indicate a possible epigenetic mechanism linked to the pathogenesis of microtia. Ear dysplasia in cases with atresia may be caused by decreased expression of miRNAs regulating apoptosis. The regulatory changes associated with TP53, PUMA, FAS, FASL, and PTEN act directly or indirectly on the apoptosis-related cascades, thereby showing that they may play an important role during development, particularly in the external ear.⁶⁴

A large proportion of the human genome (62%–75%) is transcribed into many lncRNAs. LncRNAs are transcripts that are more than 200 nucleotides in length and lack long open reading frames.⁶⁵ They regulate gene expression at a transcriptional and post-transcriptional level and are also engaged in developing various human diseases. In multicellular organisms, lncRNAs have a role in silencing the expression of heritable alleles and maintaining epigenetic traits, which involve cellular differentiation and normal development. Zhang et al⁶⁶ found that many lncRNAs and mRNAs are notably altered when comparing unaffected and affected auricular cartilage tissue. A total of 180 differentially expressed lncRNAs and associated signaling pathways may be responsible for the pathogenesis of microtia.

However, the role of noncoding RNAs in the pathogenesis of microtia still needs more solid evidence, and further research is required.

CONCLUSIONS

Although the current genetic and epigenetic findings linked to microtia are comprehensive, they still cannot explain all cases. The relevance of many microtia-related candidate genes has yet to be verified, and the pathogenesis is unclear. More clinical data, therefore, needs to be collected and should include sporadic and familial cases, as well as further investigation and verification of the disease mechanism. Further technological development and the standardized establishment of a biobank can be useful for future studies. From the perspective of multiomics and single-cell levels, making full use of advanced detection technology to deeply explore and clarify the etiology and pathogenesis of the disease will contribute to prenatal diagnosis, preimplantation diagnosis, and genetic counseling and play a positive role in disease prevention.