Microtia is a congenital anomaly of the ear that ranges in severity from mild structural abnormalities to complete absence of the ear, and can occur as an isolated birth defect or as part of a spectrum of anomalies or a syndrome. Microtia is often associated with hearing loss and patients typically require treatment for hearing impairment and surgical ear reconstruction. The reported prevalence varies among regions, from 0.83 to 17.4 per 10,000 births and the prevalence is considered to be higher in Hispanics, Asians, Native Americans, and Andeans. The etiology of microtia and the cause of this wide variability in prevalence are poorly understood. Strong evidence supports the role of environmental and genetic causes for microtia. Although some studies have identified candidate genetic variants for microtia, no causal genetic mutation has been confirmed. The application of novel strategies in developmental biology and genetics has facilitated elucidation of mechanisms controlling craniofacial development. In this paper we review current knowledge of the epidemiology and genetics of microtia, including potential candidate genes supported by evidence from human syndromes and animal models. We also discuss the possible etiopathogenesis in light of the hypotheses formulated to date: neural crest cells disturbance, vascular disruption and altitude.

SUMMARY

Hmx1 is a homeodomain transcription factor expressed in the developing eye, peripheral ganglia, and branchial arches of avian and mammalian embryos. Recent studies have identified a loss-of-function allele at the HMX1 locus as the causative mutation in the oculo-auricular syndrome (OAS) in humans, characterized by ear and eye malformations. The mouse dumbo (dmbo) mutation, with similar effects on ear and eye development, also results from a loss-of-function mutation in the Hmx1 gene. A recessive dmbo mutation causing ear malformation in rats has been mapped to the chromosomal region containing the Hmx1 gene, but the nature of the causative allele is unknown. Here we show that dumbo rats and mice exhibit similar neonatal ear and eye phenotypes. In midgestation embryos, dumbo rats show a specific loss of Hmx1 expression in neural-crest-derived craniofacial mesenchyme (CM), whereas Hmx1 is expressed normally in retinal progenitors, sensory ganglia and in CM, which is derived from mesoderm. High-throughput resequencing of 1 Mb of rat chromosome 14 from dmbo/dmbo rats, encompassing the Hmx1 locus, reveals numerous divergences from the rat genomic reference sequence, but no coding changes in Hmx1. Fine genetic mapping narrows the dmbo critical region to an interval of ∼410 kb immediately downstream of the Hmx1 transcription unit. Further sequence analysis of this region reveals a 5777-bp deletion located ∼80 kb downstream in dmbo/dmbo rats that is not apparent in 137 other rat strains. The dmbo deletion region contains a highly conserved domain of ∼500 bp, which is a candidate distal enhancer and which exhibits a similar relationship to Hmx genes in all vertebrate species for which data are available. We conclude that the rat dumbo phenotype is likely to result from loss of function of an ultraconserved enhancer specifically regulating Hmx1 expression in neural-crest-derived CM. Dysregulation of Hmx1 expression is thus a candidate mechanism for congenital ear malformation, most cases of which remain unexplained.

The premature fusion of the paired frontal bones results in metopic craniosynostosis (MC) and gives rise to the clinical phenotype of trigonocephaly. Deletions of chromosome 9p22.3 are well described as a cause of MC with variably penetrant midface hypoplasia. In order to identify the gene responsible for the trigonocephaly component of the 9p22.3 syndrome, a cohort of 109 patients were assessed by high-resolution arrays and MLPA for copy number variations (CNVs) involving 9p22. Five CNVs involving FREM1, all of which were de novo variants, were identified by array-based analyses. The remaining 104 patients with MC were then subjected to targeted FREM1 gene re-sequencing, which identified 3 further mutant alleles, one of which was de novo. Consistent with a pathogenic role, mouse Frem1 mRNA and protein expression was demonstrated in the metopic suture as well as in the pericranium and dura mater. Micro-computed tomography based analyses of the mouse posterior frontal (PF) suture, the human metopic suture equivalent, revealed advanced fusion in all mice homozygous for either of two different Frem1 mutant alleles, while heterozygotes exhibited variably penetrant PF suture anomalies. Gene dosage-related penetrance of midfacial hypoplasia was also evident in the Frem1 mutants. These data suggest that CNVs and mutations involving FREM1 can be identified in a significant percentage of people with MC with or without midface hypoplasia. Furthermore, we present Frem1 mutant mice as the first bona fide mouse model of human metopic craniosynostosis and a new model for midfacial hypoplasia.

Author Summary

Although twin and family studies have shown that genes play a critical role in the timing of fusion of skull bones, the identification of specific genes that may be involved has remained somewhat elusive except in the case of the dominantly inherited craniosynostosis syndromes. Metopic craniosynostosis (MC), the early fusion of the forehead (frontal) bones, accounts for 5%–15% of all craniosynostosis cases. This premature fusion of the frontal bones results in a characteristically altered skull shape, termed trigonocephaly, that usually requires surgical correction. Remarkably, the cause of the majority of cases of MC remains unknown (idiopathic). Here, we report genetic variants involving chromosome 9 which involve and interrupt the structure of the FREM1 gene in a large cohort of patients presenting with unisutural metopic craniosynostosis. Micro-computed tomographic (microCT) imaging and quantitative analysis of skull shape reveal both premature fusion of the PF suture (metopic equivalent) and also changes in frontal bone shape supportive of a role for Frem1 in regulation of the metopic suture. Taken together with Frem1 gene and protein expression findings, these data indicate that mutations in FREM1 can give rise to metopic craniosynostosis.

Recent studies have shown that exposure to some nutritional supplements and chemicals in utero can affect the epigenome of the developing mouse embryo, resulting in adult disease. Our hypothesis is that epigenetics is also involved in the gestational programming of adult phenotype by alcohol. We have developed a model of gestational ethanol exposure in the mouse based on maternal ad libitum ingestion of 10% (v/v) ethanol between gestational days 0.5–8.5 and observed changes in the expression of an epigenetically-sensitive allele, Agouti viable yellow (Avy), in the offspring. We found that exposure to ethanol increases the probability of transcriptional silencing at this locus, resulting in more mice with an agouti-colored coat. As expected, transcriptional silencing correlated with hypermethylation at Avy. This demonstrates, for the first time, that ethanol can affect adult phenotype by altering the epigenotype of the early embryo. Interestingly, we also detected postnatal growth restriction and craniofacial dysmorphology reminiscent of fetal alcohol syndrome, in congenic a/a siblings of the Avy mice. These findings suggest that moderate ethanol exposure in utero is capable of inducing changes in the expression of genes other than Avy, a conclusion supported by our genome-wide analysis of gene expression in these mice. In addition, offspring of female mice given free access to 10% (v/v) ethanol for four days per week for ten weeks prior to conception also showed increased transcriptional silencing of the Avy allele. Our work raises the possibility of a role for epigenetics in the etiology of fetal alcohol spectrum disorders, and it provides a mouse model that will be a useful resource in the continued efforts to understand the consequences of gestational alcohol exposure at the molecular level.

Author Summary

In humans it has been known for some time that exposure to environmental insults during pregnancy can harm a developing fetus and have life-long effects on the individual's health. A well known example is fetal alcohol syndrome, where the children of mothers that consume large amounts of alcohol during pregnancy exhibit growth retardation, changes to the shape and size of the skull, and central nervous system defects. At present the molecular events underlying fetal alcohol syndrome are unknown. We have developed a model of alcohol exposure in the mouse, in which the genetics and the environment can be strictly controlled. We find that chronic exposure of the fetus to a physiologically relevant amount of alcohol during the first half of pregnancy results in epigenetic changes at a sensitive reporter gene and produces fetal alcohol syndrome-like features in some mice. Our model is a useful tool to study the underlying causes of fetal alcohol syndrome, and our work raises the interesting possibility that the long-term physical effects of alcohol exposure during pregnancy are mediated by epigenetic changes established in the fetus and then faithfully remembered for a lifetime. In the future, such epigenetic changes could be used as markers for the preclinical diagnosis and treatment of fetal alcohol spectrum disorders.

Mu opioid receptor (OPRM1) gene variants, particularly the common A118G single nucleotide polymorphism (SNP), are among the most frequently studied candidate genes associated with opioid dependence. However, despite numerous case-control studies and meta-analyses, no definitive conclusion has been reached regarding the association of the A118G SNP and risk of developing opioid dependence. This study aimed to resolve this discrepancy by reinvestigating the association between A118G SNP allelic, and for the first time, genotype frequencies and opioid dependence. A meta-analysis of sixteen case-control studies of opioid dependence was performed with a total of 5169 subjects. No association between the A118G allele (P = 0.23) and genotype (P = 0.34) frequencies and opioid dependence was found. However, significant heterogeneity between studies precluded highly definitive conclusions. In addition, the possibility that other OPRM1 SNPs albeit rarer may influence the risk of opioid dependence remains to be investigated at this level. Nonetheless, despite no evidence of a direct association with risk of dependence, A118G may still influence the pharmacological response to opioids impacting on an individual’s dosage requirements.

Background

The LIM-homeodomain transcription factors LHX7 and LHX6 have been implicated in palatogenesis in mice and thus may also contribute to the incidence of isolated palatal clefts and/or clefts of the lip and primary palate (CL/P) in humans. Causative mutations in the transcription factor IRF6 have also been identified in two allelic CL/P syndromes and common polymorphisms in the same gene are significantly associated with non-syndromal CL/P in different populations.

Results

Here we report the isolation of chick orthologues of LHX7, LHX6 and IRF6 and the first characterisation of their profiles of expression during morphogenesis of the midface with emphasis on the period around formation of the primary palate. LHX7 and LHX6 expression was restricted to the ectomesenchyme immediately underlying the ectoderm of the maxillary and mandibular primordia as well as to the lateral globular projections of the medial nasal process, again underlying the pre-fusion primary palatal epithelia. In contrast, IRF6 expression was restricted to surface epithelia, with elevated levels around the frontonasal process, the maxillary primordia, and the nasal pits. Elsewhere, high expression was also evident in the egg tooth primordium and in the apical ectodermal ridge of the developing limbs.

Conclusion

The restricted expression of both LHX genes and IRF6 in the facial primordia suggests roles for these gene products in promoting directed outgrowth and fusion of the primary palate. The manipulability, minimal cost and susceptibility of chicks to CL/P will enable more detailed investigations into how perturbations of IRF6, LHX6 and LHX7 contribute to common orofacial clefts.

Background

Patients with Opitz GBBB syndrome present with a variable array of developmental defects including craniofacial, cardiac, and genital anomalies. Mutations in the X-linked MID1 gene, which encodes a microtubule-binding protein, have been found in ~50% of Opitz GBBB syndrome patients consistent with the genetically heterogeneous nature of the disorder. A protein highly related to MID1, called MID2, has also been described that similarly associates with microtubules.

Results

To identify protein partners of MID1 and MID2 we undertook two separate yeast two-hybrid screens. Using this system we identified Alpha 4, a regulatory subunit of PP2-type phosphatases and a key component of the rapamycin-sensitive signaling pathway, as a strong interactor of both proteins. Analysis of domain-specific deletions has shown that the B-boxes of both MID1 and MID2 mediate the interaction with Alpha 4, the first demonstration in an RBCC protein of a specific role for the B-box region. In addition, we show that the MID1/2 coiled-coil motifs mediate both homo- and hetero-dimerisation, and that dimerisation is a prerequisite for association of the MID-Alpha 4 complex with microtubules.

Conclusions

Our findings not only implicate Alpha 4 in the pathogenesis of Opitz GBBB syndrome but also support our earlier hypothesis that MID2 is a modifier of the X-linked phenotype. Of further note is the observation that Alpha 4 maps to Xq13 within the region showing linkage to FG (Opitz-Kaveggia) syndrome. Overlap in the clinical features of FG and Opitz GBBB syndromes warrants investigation of Alpha 4 as a candidate for causing FG syndrome.

We report the development and optimization of reagents for in-solution, hybridization-based capture of the mouse exome. By validating this approach in a multiple inbred strains and in novel mutant strains, we show that whole exome sequencing is a robust approach for discovery of putative mutations, irrespective of strain background. We found strong candidate mutations for the majority of mutant exomes sequenced, including new models of orofacial clefting, urogenital dysmorphology, kyphosis and autoimmune hepatitis.

An extended ENU screen for modifiers of transgene variegation identified four new modifiers, MommeD7-D10.

Background

Some years ago we established an N-ethyl-N-nitrosourea screen for modifiers of transgene variegation in the mouse and a preliminary description of the first six mutant lines, named MommeD1-D6, has been published. We have reported the underlying genes in three cases: MommeD1 is a mutation in SMC hinge domain containing 1 (Smchd1), a novel modifier of epigenetic gene silencing; MommeD2 is a mutation in DNA methyltransferase 1 (Dnmt1); and MommeD4 is a mutation in Smarca 5 (Snf2h), a known chromatin remodeler. The identification of Dnmt1 and Smarca5 attest to the effectiveness of the screen design.

Results

We have now extended the screen and have identified four new modifiers, MommeD7-D10. Here we show that all ten MommeDs link to unique sites in the genome, that homozygosity for the mutations is associated with severe developmental abnormalities and that heterozygosity results in phenotypic abnormalities and reduced reproductive fitness in some cases. In addition, we have now identified the underlying genes for MommeD5 and MommeD10. MommeD5 is a mutation in Hdac1, which encodes histone deacetylase 1, and MommeD10 is a mutation in Baz1b (also known as Williams syndrome transcription factor), which encodes a transcription factor containing a PHD-type zinc finger and a bromodomain. We show that reduction in the level of Baz1b in the mouse results in craniofacial features reminiscent of Williams syndrome.

Conclusions

These results demonstrate the importance of dosage-dependent epigenetic reprogramming in the development of the embryo and the power of the screen to provide mouse models to study this process.