Background Otocephaly or dysgnathia complex is characterised by mandibular hypoplasia/agenesis, ear anomalies, microstomia, and microglossia; the molecular basis of this developmental defect is largely unknown in humans.
Methods and results This study reports a large family in which two cousins with micro/anophthalmia each gave birth to at least one child with otocephaly, suggesting a genetic relationship between anophthalmia and otocephaly. OTX2, a known microphthalmia locus, was screened in this family and a frameshifting mutation was found. The study subsequently identified in one unrelated otocephalic patient a sporadic OTX2 mutation. Because OTX2 mutations may not be sufficient to cause otocephaly, the study assayed the potential of otx2 to modify craniofacial phenotypes in the context of known otocephaly gene suppression in vivo. It was found that otx2 can interact genetically with pgap1, prrx1, and msx1 to exacerbate mandibular and midline defects during zebrafish development. However, sequencing of these loci in the OTX2-positive families did not unearth likely pathogenic lesions, suggesting further genetic heterogeneity and complexity.
Conclusion Identification of OTX2 involvement in otocephaly/dysgnathia in humans, even if loss of function mutations at this locus does not sufficiently explain the complex anatomical defects of these patients, suggests the requirement for a second genetic hit. Consistent with this notion, trans suppression of otx2 and other developmentally related genes recapitulate aspects of the otocephaly phenotype in zebrafish. This study highlights the combined utility of genetics and functional approaches to dissect both the regulatory pathways that govern craniofacial development and the genetics of this disease group.
- clinical genetics
- molecular genetics
- visual development
- genetic screening/counselling
- congenital heart disease
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- clinical genetics
- molecular genetics
- visual development
- genetic screening/counselling
- congenital heart disease
Otocephaly-dysgnathia (also known as otocephaly, agnathia-holoprosencephaly, dysgnathia complex) (OMIM #202650) is characterised by mandibular hypoplasia or agenesis, ventromedial auricular malposition (melotia or synotia), microstomia, and oroglossal hypoplasia or aglossia. The mesenchyme-forming neural crest is affected, resulting in abnormal derivatives of the caudal portion of the first branchial arch.1 Additional malformations can be associated with this condition, including, but not limited to, failure of development of the prosencephalon, resulting in midline defects as severe as alobar hemispheres or cyclopia, anophthalmia, microphthalmia, pituitary hypoplasia, situs inversus, pulmonary hypoplasia, and limb malformations.
Fewer than 150 cases have been documented, with an incidence estimated to be 1 per 70 000 births.2 Two families have been reported with more than one affected member.3 4 In one example of a genomic rearrangement, Pauli et al reported two stillborn female infants with agnathia-holoprosencephaly who harboured an unbalanced 46,XX,der18,t(6;18) (pter->p24.1 or p24.2::p11.21->qter) translocation from a parent with a balanced translocation t(6;18) (p24.1 or p24.2; p11.21).5 Karyotypes of other cases of otocephaly described in the literature have been normal, suggesting that most causative lesions are either point mutations or copy number variants that fall below detectable thresholds for karyotype or microarray. Environmental causes such as exposure to salicylates have also been suspected contributors.6
Forward genetic screens in murine models have identified numerous genes involved in otocephaly. The first otocephaly locus was identified in a screen for lethal mutations on chromosome 1,7 and the causal mutation was mapped to Pgap1 (post-glycosylphosphatidylinositol attachment to proteins 1).8 Ueda et al subsequently generated Pgap1 deficient mice and showed that they recapitulate the otocephalic phenotype and its variable penetrance and expressivity, since the phenotype of mutant pups ranged in severity from a normal face to complete lack of mouth and jaw.9 Loss of two other murine genes has also been shown to cause otocephaly-agnathia, in each case in a context dependent fashion. On a C57BL/6 genetic background, loss of the twisted gastrulation gene 1 (Twsg1−/−) results in anomalies of the first branchial arch leading to agnathia, as well as forebrain abnormalities.10 Finally, chimeric Otx2 heterozygous knockout mice also display an otocephalic phenotype, albeit with variable penetrance and expressivity attributable in part to genetic background.11
In humans, molecular defects leading to otocephaly are largely unknown. Recently, missense mutations in PRRX1 (paired-related homoeobox gene 1) were identified in two sporadic cases of otocephaly.12 13
OTX2 mutations have been observed in patients with isolated severe ocular and pituitary malformations.14 15 Here, we report the identification of a deleterious mutation in OTX2 in a large French family in which variable expressivity extends from micro/anophthalmia to otocephaly and is inherited in a dominant manner among four generations. We subsequently performed molecular screening of OTX2 in nine additional, non-related otocephalic cases and identified a second sibship with an OTX2 mutation. Despite OTX2 being a known microphthalmia locus, our data suggested that this gene might also be necessary but not sufficient in some families with otocephaly. To dissect this apparently complex genetic model, we suppressed otx2 during zebrafish development and determined that: (1) otx2 is also necessary for correct mandible formation; and (2) otx2 can interact genetically with other loci to modulate the severity of mandibular malformations. Taken together, our data support a causal role of OTX2 in otocephaly in humans but indicate that other genetic factors are likely necessary for the manifestation of the otocephalic phenotype.
Subjects, materials and methods
One family and nine sporadic otocephaly patients were included in this study. Among the sporadic patients, four have been reported previously elsewhere.16–19 Informed consents with appropriate ethics review committee approvals were obtained. DNA was extracted from blood or fresh tissue for the family, three sporadic patients, and asymptomatic parents of an otocephalic patient; DNA was extracted from paraffin embedded tissue for the remaining patients. Following DNA extraction from paraffin embedded blocks, whole genome amplification was performed using standard procedures (Sigma Aldrich, Lyon, France).
Patient descriptions including medical history, family history, physical examinations, autopsy reports, and radiological studies were obtained. Except for one family in which some members display micro/anophthalmia, all patients were diagnosed as sporadic cases of otocephaly-dysgnathia syndrome.
Complete clinical data for the family and the patient sharing the OTX2 mutations are described in the supplementary material. Briefly, in family A (figure 1A), 17 members display micro/anophthalmia (supplementary figure 1) segregating with an autosomal, dominant inheritance and sometimes associated with a variable degree of intellectual disability (moderate to severe), three patients were diagnosed as otocephalic (figure 1A), and one patient displayed clinical features overlapping both micro/anophthalmia and otocephaly, which we consider to be an intermediate phenotype (figure 1B and supplementary figure 2). The second otocephalic patient (figure 1B) harbouring an OTX2 mutation was a sporadic case (figure 1A) with no familial history of ophthalmic or mandibular malformations.
Candidate gene analysis
We sequenced OTX2 in all index cases and extended our sequencing to relatives when a change of interest was identified. Since half of the DNAs were extracted from paraffin embedded blocks, primer pairs were designed to amplify PCR products <250 bases. Patients negative for OTX2 coding and flanking splice site mutations were also screened for ALX4, MSX1, PGAP1, PRRX1, and TWSG1 mutations when the amount of DNA allowed such analyses (four out of eight patients). In addition, the same loci were sequenced in the two families with OTX2 mutations in search of candidate modifier alleles. Primers and PCR conditions used are summarised in supplementary table 1. Both DNA strands were sequenced using the Big Dye Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems, Courtaboeuf, France). GenBank accession numbers were NM_021728.2 (OTX2), NM_024989.3 (PGAP1), NM_020648.5 (TWSG1), NM_021926.3 (ALX4), and NM_002448.3 (MSX1).
Zebrafish embryo manipulation and genetic interaction studies
Splice blocking morpholinos (MOs) targeting otx2, pgap1, prrx1a and prrx1b, and a translation blocking morpholino targeting msx1 (Gene Tools, Philomath, Oregon, USA) were diluted to appropriate concentrations with sterile, nuclease-free water (3 or 9 ng/nl for each MO for dose response; 3 ng/nl for genetic interaction studies) and injected into wild-type zebrafish embryos collected from natural matings at the 1–2 cell stage according to standard procedures. Embryos were reared at 28°C in 1-phenyl-2-thiourea beginning at 24 h post-fertilisation until harvest at 5 days post-fertilisation (dpf). All experiments (n=66–75 embryos/injection) were repeated twice. To assess cartilaginous craniofacial structures, embryos were anaesthetised with tricaine, fixed overnight in 4% paraformaldehyde, and stained overnight in Alcian blue solution (0.1% Alcian blue, 70% ethanol, 1% HCl). Embryos were cleared with acidic ethanol (70% ethanol, 5% HCl) for 4 h, dehydrated in 100% ethanol, and imaged in glycerol. All images (live and Alcian blue stained whole embryos) were acquired on a Nikon AZ100 stereoscope at 6× magnification using Nikon NIS Elements software. To assess knockdown efficiency of each splice blocking morpholino, we harvested total RNA from injected batches of 25 embryos using Trizol (Invitrogen, Grand Island, NY, USA) according to the manufacturer's instructions. Oligo-dT-primed cDNA was then synthesised using SuperScriptIII reverse transcriptase (Invitrogen) and cDNA was subsequently PCR amplified using primers flanking the MO target sites for each of otx2, pgap1, prrx1a and prrx1b.
OTX2 is a candidate gene for otocephaly-agnathia
In family A, we identified a c.316delC mutation in exon 3 of OTX2 (figure 2), a deletion predicted to result in a frameshift, p.Gln106AsnfsX11. The truncated protein is predicted to terminate at the end of the homeodomain within the glutamine stretch (figure 2). Since otocephalic patients IV-1 and IV-2 died about 20 years ago, their DNAs were unavailable to investigate their OTX2 mutational burden. However, their mother (III-2) was found to have the mutation, suggesting that her two otocephalic offspring IV-1 and IV-2 likely inherited this mutation from her. Individual IV-7, with ocular abnormalities, also carried the mutation, suggesting that his otocephalic sibling IV-6 may have had the mutation, although we were unable to obtain a sample for testing from IV-6 or from their affected father III-9. Patient IV-3, with clinical features overlapping both micro/anophthalmia and otocephaly, did share the familial OTX2 mutation.
In family B (figure 1A), we screened OTX2 in a female patient with otocephaly (figure 1B) and detected a c.130delC mutation in exon 2 (figure 2). This deletion is predicted to result in a frameshift, p.Arg44GlyfsX15; if translated, the truncated protein would terminate before the OTX2 homeodomain (figure 2). This patient was a sporadic case with no family history of ophthalmic or mandibular malformations. Parental DNA analysis showed that the OTX2 mutation appears de novo. No OTX2 mutation was identified in the 8 remaining otocephalic patients. These results are summarised in supplementary table 2.
Candidate gene screening of ALX4, MSX1, PGAP1, PRRX1, and TWSG1 in OTX2 mutation-negative otocephaly samples
To explore further the genetic basis of otocephaly and overlapping phenotypes in the OTX2 mutation-negative samples in our cohort, we conducted molecular analysis of five additional candidate genes known to play a role in otocephaly malformations in vertebrates (ALX4, MSX1, PGAP1, PRRX1, and TWSG1; possible for four out of the eight remaining patients with DNA of sufficient quality). One patient displayed two heterozygous PGAP1 intronic variations (c.1-115C>T and c.927+31A>G), while another one displayed two heterozygous variations in MSX1 (c.119C>G [p.Ala40Gly] and c.*+6C>T). More variants were identified in ALX4: c.63C>T [p.Tyr21Tyr], c.104G>C, [p.Arg35Thr], c.304C>T (p.Pro102Ser), c.621A>G [p.Ser207Ser], c.1074C>T [p.His358Gln], and c.*228C>T. All sequence variations are common single nucleotide polymorphisms (SNPs) referenced in dbSNP. These variants are thus unlikely to cause otocephaly. No PRRX1 or TWSG1 variation was identified. Patient genotypes are summarised in supplementary table 2.
otx2 interacts with other otocephaly loci in an in vivo developmental model
Because of the observed phenotypic variability observed among affected individuals in our families, we hypothesised that lesions in additional loci may interact with OTX2 to cause otocephaly—an hypothesis consistent with the reported background dependent variable penetrance and expressivity of murine otocephaly mutations.11 20 We have shown previously that the zebrafish is a useful model to dissect epistasis contributing to variable phenotypes observed in human developmental disorders.21–23 Therefore, we investigated the potential for otx2 to modulate specific otocephaly endophenotypes or severity by suppressing otx2 in a sensitised, physiologically relevant context in the developing zebrafish embryo. First, we identified the single zebrafish ortholog of OTX2 (91% identity; 94% similarity vs human) and suppressed it transiently by microinjection of an antisense MO targeting the splice donor sequence of Danio. rerio otx2 exon 2 in batches of wild-type (wt) embryos at the one-to-two cell stage. At 5 dpf, we scored embryos for craniofacial phenotypes relevant to otocephaly and we observed mild microphthalmia and shortening of the pharyngeal skeleton that increased in penetrance in a dose dependent manner (figure 3A, supplementary figure 3J). Notably, Alcian blue staining of 5dpf embryos revealed the presence of all pharyngeal components, but distinct defects in comparison to controls. These data were consistent with the specific and robust targeting of the MO as indicated by RT-PCR of cDNA generated from total RNA harvested from otx2 morphants (supplementary figure 3A,E).
Next, we asked whether otx2 could exacerbate the craniofacial phenotypes that result from loss of the known otocephaly loci, PGAP1 and PRRX1, and an additional locus implicated in mandibular development, MSX1. First, we identified the zebrafish orthologs of each protein; PGAP1 (one copy; 43% identity, 64% similarity vs human), PRRX1 (two copies; a and b each, 85% identity, 91% similarity vs human), and MSX1 (one copy; 61% identity, 68% similarity vs human) could each be suppressed efficiently in developing zebrafish embryos subsequent to injection of splice blocking (pgap1, prrx1a and prrx1b) or translation blocking (msx1) MOs (supplementary figure 3B–D, F–J). Scoring of zebrafish larva at 5 dpf revealed that knockdown of pgap1 resulted in mild microphthalmia, fusion of the eyes at the midline, and protrusion of the mandible (figure 3A,D); prrx1a/b double morphants and msx1 morphants displayed reduced eye size and anterior-posterior shortening of jaw structures (figure 3A,D). Similar to otx2, these abnormalities in the pharyngeal skeleton were dose dependent (supplementary figure 3J). Next, to test otx2 genetic interaction with pgap1, we injected subeffective doses of each MO (3 ng each of otx2 and/or pgap1) either alone or in a pairwise fashion into wt zebrafish embryos at the 1–2 cell stage and scored them live for craniofacial defects at 5 dpf. Whereas we observed a modest percentage of abnormal embryos for otx2 and pgap1 individually (2% and 26%, respectively; n=66–74 embryos/injection, repeated at least twice with masked scoring), the combined effect resulted in a synergistic exacerbation of phenotypes when compared to either MO alone. Pairwise suppression resulted in increased mortality and a new severe class of embryos that displayed severe microphthalmia, eye fusion along the midline, and severe disorganisation of mandibular cartilage as indicated by Alcian blue staining (figure 3A,B,D). Suppression of otx2 also exacerbated the effects of prrx1a/b knockdown. Through comparisons of subeffective MO injection doses targeting either otx2 and/or prrx1a/b (3 ng of each MO), we saw a pronounced increase in embryos with craniofacial defects and a severe class of embryos in the otx2/prrx1a/b morphant injection batches (2%, 4%, and 32% affected embryos for otx2, prrx1a/b and otx2/prrx1a/b double morphants, respectively; n=66–74 embryos/injection; figure 3A,C,D). Similarly, pairwise interaction studies between otx2 and msx1 resulted in exacerbation of craniofacial defects in otx2/msx1 injection batches compared to either single MO alone (3%, 6%, and 22% affected embryos for otx2, msx1 and otx2/msx1 double morphants respectively; n=66–75 embryos/injection; figure 3A,C,D). Together, these results indicate that suppression of otx2, in combination with loss of function of other loci contributing to otocephaly phenotypes, can modulate phenotypic severity in the manifestation of craniofacial malformations.
ALX4, MSX1, PGAP1, PRRX1, and TWSG1 as candidate genetic interactors of OTX2
Informed by the in vivo functional studies, which indicated the potential for OTX2 to modulate otocephaly phenotypes, we returned to the patient cohorts for further mutational analysis. We screened the five candidate genes listed above (ALX4, MSX1, PGAP1, PRRX1, and TWSG1) in available family members from each of the two families bearing pathogenic OTX2 mutations. Variants identified among ALX4, MSX1, PGAP1, PRRX1, and TWSG1 are described below and summarised in supplementary table 2. First, in PRRX1, we detected no novel changes. In ALX4, PGAP1, TWSG1, and MSX1, only known SNPs and all but two (c.778-11G>A in ALX4, and c.906T>C [p.Leu302Leu] in PGAP1) shared by micro/anophthalmic relatives were identified. The ALX4 c.778-11G>A variation is not predicted to alter splicing by in silico analysis and the PGAP1 c.906T>C variation is a neutral variation with no amino acid modification and no predicted effect on splicing by in silico analysis. Variations identified among the screened genes are thus unlikely to exert a modifier effect leading to otocephaly.
Otocephaly/agnathia is the most severe known developmental defect of the mandible. Three genes (Otx2, Pgap1, Twsg1) cause otocephaly when inactivated in mice, but the molecular defects underlying this severe malformation are still largely unknown in humans. Recently, missense mutations in PRRX1, encoding a transcriptional co-activator, was identified in two cases of otocephaly.12 13 Functional studies indicated that these mutations decrease the ability for the mutant protein to regulate the tenascin-C gene promoter, and thus, these mutations were considered as deleterious. Prrx1 null mice display cleft palate and mild hypoplasia of both the mandible and the zeugopodal bones of the limbs.24 These are the only cases reported with a plausible molecular explanation of otocephaly. However, we cannot exclude the possibility that additional mutational burden was required to manifest the severe craniofacial phenotype.
In this report, we demonstrated that OTX2 mutations contribute to this malformation in humans. We identified an OTX2 mutation in a large family where an autosomal dominant form of micro/anophthalmia was present. Two microphthalmic cousins of this family each gave birth to at least one child with otocephalic features. Since these otocephalic patients died prenatally or shortly after birth 20 years ago, it is not possible to establish whether they shared the familial mutation. However, we were able to show that their affected parent harboured the familial OTX2 null mutation. Additionally, in the same family, we identified the familial OTX2 mutation in a fetus displaying ocular (microphthalmia with retinal dysplasia and absence of the anterior chamber) and mandibular (severe micrognathia) features.
To confirm the role of OTX2 mutations in otocephaly, we screened an additional nine unrelated otocephalic patients for OTX2 mutations. In one patient, we found a de novo frameshifting OTX2 mutation, thus confirming the implication of this gene in otocephaly. In our series, an OTX2 mutation was identified in 2/10 (20%) probands, and no other OTX2 mutations could be found in the remainder of our cohort. Despite the possibility that we missed mutations by direct sequencing (exonic rearrangements, splicing mutation located far from the coding sequence, or mutations in regulatory regions), this result supports a probable genetic heterogeneity for otocephaly.
We performed additional molecular analysis for four of the eight patients mutation-negative for OTX2, focusing on plausible functional candidates (ALX4, MSX1, PGAP1, PRRX1, and TWSG1). Alx4, a gene involved in skull defects, has previously been proposed to be a modifier of the otocephalic phenotype in Otx2 heterozygous mutant mice.20 MSX1 is a gene involved in mandibular embryonic development,25 and its expression is regulated directly or indirectly by TWSG126 and OTX2.27 In addition, MSX1 binds a distant non-coding regulatory element of SOX9 which, when mutated, leads to Pierre-Robin sequence, a less severe human mandibular phenotype.28 Pgap1 and Twsg1 cause otocephaly when inactivated in mice.9 10 Finally, a mutation in PRRX1 was previously identified in two otocephalic patients.12 13 No deleterious mutations were identified in any of these genes in the OTX2 mutation-negative patients. However, this particular cohort is too small to rule out their possible contribution to human otocephalic phenotypes.
The major phenotype described previously in patients with OTX2 mutations is microphthalmia/anophthalmia associated with extra-ocular defects such as brain malformations, pituitary abnormalities, short stature, and mental retardation.15 OTX2 mutations identified so far are listed in supplementary table 3, and represented in figure 2B. Phenotypic variability and incomplete penetrance have also been documented.14 15 No obvious phenotype/genotype correlations for the single gene OTX2 can be made. First, several micro/anophthalmic patients harbour frameshifting or nonsense mutations located in the same region of OTX2 (figure 2B), indicating that haploinsufficiency at this locus is likely insufficient to cause otocephaly. Second, our epistatic analysis shows overt genetic interactions with other genes known to be required for mandibular formation in humans and/or rodents. Together, these data suggest that genetic interactions as well as the position/type of mutations at OTX2 likely drive phenotypic expressivity.
The otocephaly-dysgnathia spectrum ranges from isolated mandibular involvement (dysgnathia or agnathia) to a broader spectrum of malformations including dysgnathia, holoprosencephaly, situs inversus, and visceral anomalies.29 Of note, the patient with OTX2 mutations and otocephaly whom we could examine (family B, patient II-1) was not affected by features of holoprosencephaly, situs inversus, or by visceral malformations. In one patient with an intermediate phenotype (family A, patient IV-3), thymic hyperplasia, 11 rib pairs, and micropenis were also associated with the otocephaly.
Discrete OTX2 expression in the mammalian forebrain and retinal anlage is preceded and accompanied by transcription in the anterior mesendoderm and pharyngeal endoderm.30 This region of pharyngeal endoderm is critical for the induction and orientation of facial skeletal elements derived from cephalic neural crest cells.31 We speculate that the effect of OTX2 mutations on eye formation would be direct within the neuroepithelial component, but indirect, via mis-specification of the rostral pharyngeal endoderm, on the mandibular portion of the first branchial arch.
Consistent with observations in OTX2 null mice, the mandibular phenotype associated with OTX2 mutation in humans is highly variable, ranging from absence of developmental defect (most of the family A members), to micrognathia (family A, patient IV-3), to agnathia (family B), to severe otocephaly (family A, patients IV-1, IV-2, and IV-6). Phenotypic variation has been attributed to modifier genes, environmental variations, and stochastic effects. To understand the phenotypic variability observed between the two families with OTX2 mutations, we hypothesised that pathogenic alleles at epistatic loci may be involved in the otocephalic phenotype for patients bearing an OTX2 mutation. In vivo modelling experiments in zebrafish showed that suppression of three genes in concert with otx2, (pgap1, prrx1a/b, and msx1) lead to exacerbated craniofacial defects far exceeding defects of each gene alone, suggesting that the combinatorial effect of additional molecular lesions in the genome may explain the phenotypic variability associated with OTX2 mutations. In our families, known otocephaly-causing/contributing genes were mutation negative for the candidates screened, suggesting that the otocephaly phenotype is likely subject to additional genetic heterogeneity. Whole exome/genome sequencing of these and other families under a ‘two-hit’ hypothesis, coupled to epistatic analysis in zebrafish or other suitable model organisms, is likely to identify such alleles and illuminate the genetic architecture of this complex disorder.
The authors thank the families for their participation and the following physicians and scientists for their assistance: Petra Reinecke at University Hospital, Duesseldorf, Germany; Didier Lacombe at CHU Pellegrin-Enfants, Bordeaux Cedex, France; Marion Gerard at Robert Debré Hospital, Paris, France; Christine Peres, Annaïck Desmaison, Matthias Macé and Stanislas Faguer at Inserm U563, Toulouse, France, and the French Foetopathology Society (SOFFOET). NK is a Distinguished George W. Brumley Professor.
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NC, SS contributed equally to this work.
Funding This work was supported by grants from the Clinical Research Hospital Program from the French Ministry of Health (PHRC 09 109 01), Retina France, and from National Institutes of Health (5 R01 DE13849 and -09 S1).
Competing interests None.
Patient consent Obtained.
Ethics approval Ethics approval was provided by the Comité de Protection des Personnes Sud-Ouest et Outre-Mer II.
Provenance and peer review Not commissioned; externally peer reviewed.
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