Article Text

Original article
Heterogeneity of mutational mechanisms and modes of inheritance in auriculocondylar syndrome
  1. Christopher T Gordon1,
  2. Alice Vuillot1,
  3. Sandrine Marlin2,
  4. Erica Gerkes3,
  5. Alex Henderson4,
  6. Adila AlKindy5,
  7. Muriel Holder-Espinasse6,
  8. Sarah S Park7,
  9. Asma Omarjee1,
  10. Mateo Sanchis-Borja1,
  11. Eya Ben Bdira1,
  12. Myriam Oufadem1,
  13. Birgit Sikkema-Raddatz3,
  14. Alison Stewart8,
  15. Rodger Palmer9,
  16. Ruth McGowan10,
  17. Florence Petit6,
  18. Bruno Delobel11,
  19. Michael R Speicher12,
  20. Paul Aurora13,
  21. David Kilner13,
  22. Philippe Pellerin14,
  23. Marie Simon15,
  24. Jean-Paul Bonnefont15,
  25. Edward S Tobias16,
  26. Sixto García-Miñaúr17,
  27. Maria Bitner-Glindzicz18,
  28. Pernille Lindholm19,
  29. Brigitte A Meijer20,
  30. Véronique Abadie21,
  31. Françoise Denoyelle22,
  32. Marie-Paule Vazquez23,24,25,
  33. Christa Rotky-Fast26,
  34. Vincent Couloigner27,
  35. Sébastien Pierrot27,
  36. Yves Manach27,
  37. Sylvain Breton28,29,
  38. Yvonne M C Hendriks30,
  39. Arnold Munnich1,15,
  40. Linda Jakobsen19,
  41. Peter Kroisel12,
  42. Angela Lin31,
  43. Leonard B Kaban32,
  44. Lina Basel-Vanagaite33,34,35,
  45. Louise Wilson9,
  46. Michael L Cunningham7,36,
  47. Stanislas Lyonnet1,15,
  48. Jeanne Amiel1,15
  1. 1INSERM U781, Hôpital Necker-Enfants Malades and Université Paris Descartes–Sorbonne Paris Cité, Institut Imagine, Paris, France
  2. 2Centre de référence des surdités génétiques, Service de génétique médicale, Hôpital d'Enfants Armand-Trousseau AP-HP, Paris, France
  3. 3Department of Genetics, University of Groningen, University Medical Center Groningen, Groningen, the Netherlands
  4. 4Northern Genetics Service, Newcastle upon Tyne Hospitals NHS Foundation Trust, Newcastle Upon Tyne, UK
  5. 5Department of Genetics, Sultan Qaboos University Hospital, Muscat, Oman
  6. 6Service de Génétique Clinique, Hôpital Jeanne de Flandre, CHRU Lille, France
  7. 7Center for Tissue and Cell Sciences, Seattle Children's Research Institute, Seattle, USA
  8. 8West Midlands Regional Genetics Service, Birmingham, UK
  9. 9North East Thames Regional Genetics Service, Great Ormond Street Hospital, London, UK
  10. 10North Scotland Regional Genetics Service, Ashgrove House, Aberdeen, UK
  11. 11Laboratoire de génétique chromosomique, Hôpital St Vincent de Paul, Lille, France
  12. 12Institute of Human Genetics, Medical University of Graz, Graz, Austria
  13. 13Respiratory Medicine, Great Ormond Street Hospital, London, UK
  14. 14Centre de Référence des Malformations Cranio-maxillo-faciales Rares, CHRU Lille, France
  15. 15AP-HP, Département de Génétique, Hôpital Necker-Enfants Malades, Paris, France
  16. 16School of Medicine, University of Glasgow, Yorkhill Hospital, Glasgow, UK
  17. 17Institute for Medical and Molecular Genetics, La Paz University Hospital, Madrid, Spain
  18. 18Clinical and Molecular Genetics, Institute of Child Health, UCL, London, UK
  19. 19Department of Plastic and Reconstructive Surgery, Copenhagen University Hospital, Rigshospitalet, Denmark
  20. 20Department of Oral and Maxillofacial Surgery, Kennemer Gasthuis, Haarlem, The Netherlands
  21. 21Service de Pédiatrie Générale, Université Paris Descartes, Hôpital Necker-Enfants Malades, Paris, France
  22. 22INSERM UMR-S587 and Pediatric Otolaryngology Department, Armand-Trousseau Children's Hospital, AP-HP, Paris 6 University, Paris, France
  23. 23Service de Chirurgie Maxillo-Faciale et Plastique, CRMR des Malformations de la Face et de la Cavité Buccale, Hôpital Necker-Enfants Malades, AP-HP, Paris, France
  24. 24Université Paris 5, UFR de Médecine Paris-Descartes, Paris, France
  25. 25Centre de Recherche des Cordeliers, UMR S 872, Paris, France
  26. 26Department of Pediatrics and Adolescence Medicine, Medical University of Graz, Graz, Austria
  27. 27Service d'ORL Pediatrique, Hôpital Necker-Enfants Malades, AP-HP, Université Paris 5, Paris, France
  28. 28AP-HP, Service d'Imagerie Pédiatrique, Hôpital Necker-Enfants Malades, Paris, France
  29. 29Laboratoire d'Anatomie, Université Paris-Descartes, Paris, France
  30. 30Department of Clinical Genetics, Free University Medical Center, Amsterdam, The Netherlands
  31. 31Genetics Unit, MassGeneral Hospital for Children, Boston, Massachusetts, USA
  32. 32Department of Oral and Maxillofacial Surgery, Massachusetts General Hospital, Boston, Massachusetts, USA
  33. 33Pediatric Genetics, Schneider Children's Medical Center of Israel and Raphael Recanati Genetics Institute, Rabin Medical Center, Beilinson Campus, Petah Tikva, Israel
  34. 34Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel;
  35. 35Felsenstein Medical Research Center, Rabin Medical Center, Petah Tikva, Israel
  36. 36Department of Pediatrics, University of Washington and Seattle Children's Hospital Craniofacial Center, Seattle, Washington, USA
  1. Correspondence to Dr Christopher T Gordon and Professor Jeanne Amiel, INSERM U781, Tour Lavoisier 2ème étage, Hôpital Necker-Enfants Malades, 149 rue de Sèvres, Paris 75015, France; chris.gordon{at}, jeanne.amiel{at}


Background Auriculocondylar syndrome (ACS) is a rare craniofacial disorder consisting of micrognathia, mandibular condyle hypoplasia and a specific malformation of the ear at the junction between the lobe and helix. Missense heterozygous mutations in the phospholipase C, β 4 (PLCB4) and guanine nucleotide binding protein (G protein), α inhibiting activity polypeptide 3 (GNAI3) genes have recently been identified in ACS patients by exome sequencing. These genes are predicted to function within the G protein-coupled endothelin receptor pathway during craniofacial development.

Results We report eight additional cases ascribed to PLCB4 or GNAI3 gene lesions, comprising six heterozygous PLCB4 missense mutations, one heterozygous GNAI3 missense mutation and one homozygous PLCB4 intragenic deletion. Certain residues represent mutational hotspots; of the total of 11 ACS PLCB4 missense mutations now described, five disrupt Arg621 and two disrupt Asp360. The narrow distribution of mutations within protein space suggests that the mutations may result in dominantly interfering proteins, rather than haploinsufficiency. The consanguineous parents of the patient with a homozygous PLCB4 deletion each harboured the heterozygous deletion, but did not present the ACS phenotype, further suggesting that ACS is not caused by PLCB4 haploinsufficiency. In addition to ACS, the patient harbouring a homozygous deletion presented with central apnoea, a phenotype that has not been previously reported in ACS patients.

Conclusions These findings indicate that ACS is not only genetically heterogeneous but also an autosomal dominant or recessive condition according to the nature of the PLCB4 gene lesion.

  • PLCB4
  • GNAI3
  • auriculocondylar syndrome
  • question mark ear
  • micrognathia

Statistics from


Auriculocondylar syndrome (ACS; MIM 602483 and 614669) is a craniofacial disorder with the core symptoms variable micrognathia, mandibular condyle hypoplasia and a specific malformation of the external ear at the junction between the lobe and helix (ranging from an indentation between the two to complete separation), known as a ‘question mark ear’ (QME). Other features of variable frequency include prominent cheeks, microstomia, palatal anomalies, glossoptosis, pre- and postauricular tags, facial asymmetry, crowded teeth and hearing loss (HL) (reviewed in Kokitsu–Nakata et al1). QME can occur as an isolated anomaly (IQME; MIM 612798). ACS is extremely rare, with less than 50 cases (including IQME) reported.1–27 A high degree of intrafamilial phenotypic variability has been reported, with several examples of incomplete penetrance.4 ,24 The first locus for ACS was mapped to a large interval on chromosome 1.2 Recently, exome sequencing of several ACS cases led to the identification of missense mutations in phospholipase C, β 4 (PLCB4) and in guanine nucleotide-binding protein (G protein), α inhibiting activity polypeptide 3 (GNAI3),24 the latter gene falling within the previously mapped interval on chromosome 1. All mutations reported thus far fall within the catalytic domain of each protein, and were predicted by Rieder et al24 to result in dominant negative (PLCB4) or gain-of-function (GNAI3) proteins. Both proteins are predicted to function downstream of endothelin receptor type A, a G protein-coupled receptor in the endothelin 1 (EDN1) signalling pathway, which is known to play a crucial role in branchial arch patterning (reviewed in Clouthier et al28). Missense mutations in the catalytic domain of plcb3 (a homologue of PLCB4) were identified in the zebrafish mutant schmerle, which displays a reduction of anterior branchial arch cartilage elements, and plcb3 was shown to interact genetically with edn1,29 supporting the involvement of PLCB4 in EDN1-regulated branchial arch development in humans.

The molecular bases of IQME are currently unknown; these cases may represent a genetically distinct entity, but given the variable expressivity that exists in PLCB4/GNAI3 mutation-positive ACS cases, mutations in these two genes may also underlie IQME. Also, it was recently proposed that a series of patients with heterogeneous disorders of the first and second branchial arches, some of which resembled mild oculoauriculovertebral spectrum (OAVS) or Goldenhar syndrome (GS; MIM 164210), could be classified within ACS.30 In this report, we tested for mutations in PLCB4 and GNAI3 in classic ACS, and overlapping phenotypes, such as IQME and OAVS/GS. Our results suggest a narrow phenotypic spectrum for PLCB4/GNAI3 mutations, with mutation-dependent modes of inheritance. Interestingly, central apnoea may be an additional feature associated with PLCB4 loss of function.


Patient classification

Patients with ACS, QME or related phenotypes were identified from the literature and by dysmorphologists, and classified into the following categories: eight cases with clinical ACS (meeting the minimal criteria of QME, ranging from a minor indentation at the helix-lobe junction to complete separation of the lobe from the helix, and micro- and/or retrognathia), three cases of IQME, five cases of atypical ACS previously reported by McGowan et al,30 six cases diagnosed as OAVS or GS, and four cases presenting non-syndromic auricular dysplasia (including microtia, bifid lobe or hypoplastic lobe) with or without mandibular dysplasia (table 1).

Table 1

Summary of key phenotypic features of patients tested for PLCB4 and GNAI3 mutations in the present report, and their respective genotypes


Sanger sequencing was performed according to standard techniques. In all patients, we sequenced PLCB4 exons 13–17 and 21–25 (coding for the catalytic domain) and all eight coding exons of GNAI3. In ACS and IQME patients in whom mutations were not identified in the PLCB4 catalytic domain or in GNAI3, we sequenced all remaining coding exons of PLCB4, the catalytic domain exons of PLCB3 (exons 10–13 and 15–18), and all coding exons of GNAQ and GNA11. Primer sequences used for amplification of PLCB4, GNAI3, PLCB3, GNAQ and GNA11 exons are listed in online supplementary table S1.

Microsatellite analysis

Amplification and analysis of microsatellites were performed by standard techniques. Primer sequences used for amplification of microsatellites are listed in online supplementary table S2. Homozygosity in the interval containing PLCB4 was tested with microsatellites D20S851 (426 kb upstream of the PLCB4 transcript variant 1 start site), D20S917 (falling within intron 2 of PLCB4) and BBS6 (1119 kb downstream of the PLCB4 transcript variant 1 start site).

Comparative genomic hybridisation

Case 8 was tested for chromosomal anomalies using a 135K comparative genomic hybridisation (CGH) whole genome array (Roche Nimblegen), with data analysed using InfoQuant Fusion software. The single probe showing reduced signal within the deleted interval is at chr20:9 392 652 (these and subsequent genomic coordinates refer to genome assembly Hg19).

Mutation modelling

Crystal structures were visualised using UCSF Chimera V.1.6. A crystal structure for PLCB4 has not been reported; we therefore modelled the residues mutated in PLCB4 to the structure of its paralogue, PLCB3. Protein Data Bank structure IDs: GNAI3, 2ODE;31 PLCB3, 3OHM.32

Web resources

Variant screening was performed using the dbSNP135 database via the UCSC browser (, and at the Exome Variant Server ( Evolutionary conservation of affected residues was assessed using Multiz at the UCSC browser. The effect of mutations on protein function was predicted using PolyPhen-2 ( Chromosomal aberrations were identified at the DECIPHER database (


In this report, we studied 27 cases (see table 1), and we identified seven new mutations in PLCB4 or GNAI3 (cases 1–7) and one novel homozygous intragenic deletion in PLCB4 (case 8). We present further phenotypic information for one case with a previously published PLCB4 mutation (case 9). No causal mutations were identified in cases 10–27. All mutations in cases 1–7 were absent from dbSNP135 and the Exome Variant Server, affected highly conserved residues, and were predicted probably damaging by PolyPhen-2. For each de novo case (cases 3, 4 and 6), analysis of at least 11 microsatellites was consistent with paternity.

Case 1 was previously reported as a familial case of IQME, affecting three generations.18 In the proband and her affected father, we identified a heterozygous missense mutation in PLCB4 on chromosome 20: c.1073A>T, p.Glu358Val (NCBI Reference Sequences: NM_000933.3 and NP_000924.3) (figure 1A). DNA was not available from the affected paternal grandfather. We identified the same mutation in the proband's sister, who presented no external auricular or mandibular anomalies, suggesting incomplete penetrance. Although no mandibular abnormalities were previously reported in this family, some degree of mandibular dysplasia may have been present in the affected grandfather (figure 1D in Shkalim et al18) and also in the father as an adult (permission was not granted to publish photographs). We have not been able to assess x-rays or scans of the mandible in this family. Catalytic domains of PLC enzymes are composed of two highly conserved subdomains: the X and Y domains (figure 5A). Glu358 falls with the X domain, and is predicted to be a calcium-binding residue within the active site (( and see below for structural modelling).

Figure 5

(A) distribution of mutations in PLCB4 and GNAI3 relative to domain structure. PLCB4 domains in grey: PH (pleckstrin homology), EF (EF hand-like), X and Y (catalytic), C2. GNAI3 domains in grey: boxes G1–G5. Mutations reported here are in red above each protein, and those reported in Rieder et al24 are in black below. (B) mapping of mutated PLCB4 amino acids to the PLCB3 crystal structure. Residues previously reported as mutated24 (Asn329, Arg621, Tyr623, Asn650) are in red, and those reported here (Glu358 and Asp360) are in magenta. Note that the numbering of these six residues refers to their position in the PLCB4 sequence, not PLCB3, but each of the six amino acids is identical at the homologous position in PLCB3. Calcium (Ca) is in white. (C) mapping of mutated amino acids to the GNAI3 crystal structure. The residue previously reported as mutated24 (Gly40) is in red, the residue mutated in case 7 of this report (Ser47) is in magenta, GDP is in white, aluminium tetrafluoride (AlF4) is in cyan and magnesium (Mg) is in green. This figure is only reproduced in colour in the online version.

Figure 1

Identification of missense mutations in PLCB4 and GNAI3 in auriculocondylar syndrome patients. A mutant or wild-type allele is denoted by a minus or plus sign, respectively. A question mark indicates that no information was available. Each proband is indicated by an asterisk. This figure is only reproduced in colour in the online version.

Case 2. A description of the craniofacial features of case 2 has been previously published.25 The authors labelled her phenotype as dysgnathia complex, and she presented with the core features of ACS including a crease between the lobe and helix, micrognathia and dysplastic temporomandibular joints upon radiography. Sequencing of PLCB4 revealed the mutation c.1078G>A, p.Asp360Asn (figures 1B and 5A). We were unable to obtain parental data for case 2. As for the residue mutated in case 1 (Glu358), Asp360 is predicted to contact the active site calcium.

Case 3. In a previous report, the phenotype of case 3 was briefly described as unilateral QME and micrognathia (case 2 in Greig et al23). This patient's ear deformity consists of a subtle notch at the junction between lobe and helix, and is therefore at the mildest end of the QME spectrum. A CT scan revealed severe bilateral condylar hypoplasia and dysmorphic ramus/condyle units. An initial sleep study indicated predominantly obstructive apnoeas with total number of central apnoea (CA)/obstructive apnoea (OA)/mixed apnoea (MA)/hypopnea (H) events=6/21/2/53. In a subsequent sleep study performed after one of several distraction surgeries, the majority of apnoeas appeared central, occurring generally during rapid eye movement (REM) sleep or after arousals, although they were not deemed to be pathologic, with total CA/OA/MA/H events=26/0/0/1. Sequencing of the proband and his parents identified a de novo mutation in the proband in PLCB4: c.1079A>T, p.Asp360Val (figures 1C and 5A). This is the same calcium-binding residue mutated in case 2 above, although a different nucleotidic change.

Case 4 has been previously described,9 and displays typical features of ACS. Postauricular tags were noted by Gerkes et al9; this feature is not frequently reported in the ACS literature, yet appears to be specific for this syndrome. We identified a de novo mutation in PLCB4 in the proband: c.1861C>T, p.Arg621Cys, falling in the Y domain of the catalytic domain (figures 1D and 5A). Supporting this de novo mutation being causative, the parents presented no physical abnormalities, and had normal orthopantograms. This is exactly the same mutation identified by Rieder et al24 in a familial ACS case (pedigree M003).

Case 5, from the UK, presented classic features of ACS, that is, QME (previously published as Figures 62c and 66a in Hunter et al33), severe micrognathia, large cheeks and microstomia (figure 2Ai–iii). She displayed over-folded helices and a large postauricular tag on her left ear. Her father has a small low-set right ear with a dysplastic and over-folded helix, without anomaly at the lobe-helix junction (figure 2Aiv–vi). An orthopantogram of the father revealed shorter than normal mandibular rami with a flattened head of the right condyle. The proband and her father harboured the PLCB4 mutation c.1862G>T, p.Arg621Leu (figures 1E and 5A). We were unable to test whether the mutation arose de novo in the father, as his parents are no longer alive, but examination of photos did not suggest that they were affected.

Figure 2

Craniofacial features of auriculocondylar syndrome patients harbouring PLCB4 or GNAI3 mutations. Ai–iii, case 5 (PLCB4 mutation Arg621Leu); Aiv–vi, case 5's father (PLCB4 mutation Arg621Leu). Bi–iii, case 6 (PLCB4 mutation Arg621His). Ci–iii, case 7 (GNAI3 mutation Ser47Arg); Civ–vi, case 7's father (GNAI3 mutation Ser47Arg). A large postauricular tag in Aiii is indicated by an arrow. The arrow in Biii indicates a large tag below the ear that may be a remnant of the ear lobe. An arrow in Cii indicates a notch in the external margin of the ear, at the junction between the helix and the lobe. This figure is only reproduced in colour in the online version.

Case 6 is from Oman. He presented with neonatal hypotonia, developmental delay, feeding difficulties and upper airway obstruction. He was diagnosed with ACS based on micrognathia (with hypoplastic mandibular ramus upon CT scan), full cheeks, microstomia, difficulties in opening the mouth and low-set QMEs (figure 2Bi–iii). A skin tag positioned anterior to the sternocleidomastoid may be a remnant of the left ear lobe completely detached from the pinna (figure 2Biii). He has had several distraction surgeries. Cardiovascular and ophthalmological examinations have been normal, and he is of normal intelligence. Sequencing of PLCB4 identified the mutation c.1862G>A, p.Arg621His (figures 1F and 5A). The parents and brother of case 6 did not harbour the mutation. This is exactly the same mutation previously found in a large ACS family (M001 in Rieder et al3 ,24). Case 6 is the fifth independent ACS case with a mutation affecting Arg621, after case 4 in this report and M003 in Rieder et al24 (both Arg621Cys), case 5 in this report (Arg621Leu), and M001 in Rieder et al24 (Arg621His).

Case 7 presented with asymmetric micrognathia, malocclusion, microstomia and a notch between the lobe and helix of the right ear (figure 2Ci–iii). She also displayed mildly hypoplastic first ribs upon x-ray. She harboured a heterozygous missense mutation in GNAI3 on chromosome 1: c.141C>A, p.Ser47Arg (NCBI Reference Sequences: NM_006496.3 and NP_006487.1) (figures 1G and 5A). The mutation was inherited from her father who had normal ears but large cheeks and possible hypoplasia of the angle of the mandible (figure 2Civ–vi). Mandibular x-rays were not available for the father. We were not able to obtain material to test for the mutation in the paternal grandparents. Several lines of evidence support the pathogenicity of this GNAI3 Ser47Arg mutation, including structural mapping (see below) and functional studies of mutations of the equivalent serine in other GTP-binding proteins (see Discussion).

Case 8 was born to healthy second-cousin parents of Indian descent, and presented with typical features of ACS, that is, micrognathia, microstomia, full cheeks, bilateral QME and a postauricular tag on the right ear (figure 3Ai). A CT scan demonstrated dysplastic condyles and shallow condylar fossae bilaterally. He has a significantly enlarged penis with descended testes, bilateral hydroceles and some hirsutism over his back. Brain MRI, echocardiogram, abdominal ultrasound, barium upper gastrointestinal (GI) study, neonatal hearing screen and ophthalmology assessment were all normal. He has ongoing swallowing problems, poor suck requiring a nasogastric tube and significant gastro-oesophageal reflux. Electromyography (EMG) sampling of the genioglossus and of the tibialis showed only chronic neurogenic changes in the tongue, suggesting a bulbar palsy. Shortly after birth, he displayed tachypnoea and a persistent oxygen requirement. Ear, nose and throat (ENT) examination showed some laryngomalacia. There was no evidence of obstruction, and mandibular elongation was not performed. Sleep studies were conducted due to recurrent profound apnoeas. In five out of six sleep studies, central apnoeas were more frequent than obstructive apnoeas: (1) CA/OA/MA/H events=106/9/0/14; (2) CA/OA/MA/H events=35/0/0/31; (3) CA/OA/MA/H events=88/1/1/5; (4) CA/OA/MA/H events=80/0/0/14; (5) CA/OA/MA/H events=291/14/1/40 and (6) CA/OA/MA/H events=39/141/29/2. Apnoeas were managed with intermittent BiPAP and ongoing supplemental oxygen. There were no obvious signs of ACS in the proband's parents (figure 3Aii, iii) nor in the maternal grandparents, who themselves are first cousins (figure 3B). Three of the mother's brothers died in childhood or young adulthood. Although they were not seen in the clinic, from photographic evidence, one appears to have had achondroplasia, the other two were not overtly dysmorphic but were intellectually deficient, and there were no visible ear malformations. Karyotype and array CGH at 0.2 Mb resolution did not reveal chromosomal anomalies in the proband. No mutations were identified in the PHOX2B coding region. Sequencing of PLCB4 identified no mutation, although we were unable to amplify exons 21–24. Suspecting a homozygous deletion, we reduced the putative deletion interval by confirming the presence of a series of amplicons in introns 20 and 24 (amplicons a–d in figure 3C). Sequencing of a proband-specific product spanning from amplicon a to b demonstrated a deletion of 4997 bp (chr20:9388282–9393278) (figure 3C, D). Three base pairs of microhomology existed at the breakpoint; this is a common copy-number variant (CNV) breakpoint signature.34 Retrospective examination of the PLCB4 locus in two independent prior array CGH experiments indicated a reduced signal for the only probe within the deleted region, consistent with homozygous loss (figure 3E). In all three RefSeq PLCB4 transcripts, the deletion is predicted to result in a frameshift and premature stop codon, and therefore, the proband should be homozygous null for PLCB4, assuming nonsense-mediated decay. Using the deletion-spanning primers for genotyping in each parent, we amplified the same deletion-spanning product of the proband, plus the wild-type product (figure 3F), indicating heterozygous intragenic deletion of PLCB4 in each parent. Accordingly, the proband was homozygous for a haplotype at the locus (defined by microsatellites D20S851, D20S917 and BBS6) that was present in each parent in the heterozygous state.

Figure 3

Identification of a homozygous deletion within PLCB4 in case 8. (A) ears of the proband (i), his mother (ii) and his father (iii). An arrow in Ai indicates a small postauricular tag protruding from behind the helix. (B) case 8 pedigree. An asterisk indicates the proband. Note, that the only individuals seen in the clinic were II8, II9, III4, III5, IV1, IV2. The presence or absence of phenotypes in other family members is based on photographic evidence or as reported by the family. (C and D) localisation of the deletion breakpoints. Amplicons that could be obtained from the proband's DNA are indicated by black triangles (exons 20 and 25, and intronic amplicons a–d), while those unamplifiable are indicated by empty triangles (exons 21–24). The amplicons are depicted beneath a UCSC browser screenshot of the three PLCB4 RefSeq transcripts. Forward and reverse primers from amplicons a and b, respectively, yielded a PCR product of 1455 bp from the proband's DNA, and the precise deletion breakpoints were obtained by sequencing this product with the amplicon b reverse primer. In D, a region of microhomology at the deletion breakpoints is underlined. Bases within the deleted region are in lowercase. (E) CGH array indicating loss of a single probe (arrow) within the deleted region. (F) PCR genotyping of the parents of case 8 using forward and reverse primers from amplicons a and b, respectively. Each parent (III4 and III5) yielded a wild-type product with expected size of 6452 bp, plus the deletion-containing product of 1455 bp. IV2, proband; C +, wild-type positive control DNA; C −, reaction without DNA; M, size marker. This figure is only reproduced in colour in the online version.

Given the evidence for central apnoea in cases 3 and 8 above, we reassessed sleep studies that were performed for three of the ACS cases in Rieder et al.24 Case S011 (GNAI3 p.Gly40Arg) in that report had a sleep study after distraction and decannulation at age 6 years, with CA/OA/MA/H events=5/14/6/0. Case S001 (PLCB4 p.Tyr623Cys) had a sleep study at age 6 years after distraction and decannulation (but later on needed the tracheostomy tube replaced), with CA/OA/MA/H events=26/19/0/0. Following successful distraction and decannulation, case S008 (GNAI3 p.Gly40Arg) had a sleep study with CA/OA/MA/H events=0/2/0/25.

All ACS-associated PLCB4 point mutations reported here and in Rieder et al24 are missense mutations clustered in the catalytic domain, suggesting that they may function as dominant negatives rather than haploinsufficient alleles. This is consistent with the absence of an ACS phenotype in the parents of case 8, who each carried a heterozygous deletion. To provide further support for this, we assessed the craniofacial phenotypes of patients with chromosomal aberrations disrupting PLCB4, entered in the DECIPHER database. DECIPHER patient 253 449 harbours a deletion (likely heterozygous, based on mean ratio of −1) at chr20:8 509 979–9 822 641, containing four genes, including PLCB4 in its entirety, and an adjacent inverted duplication (chr20:126 054–8 131 005). This patient displayed no facial features suggestive of ACS. DECIPHER patient 1692 harbours a deletion (likely heterozygous, based on mean ratio of −0.7) at chr20:9 249 717–12 702 585, which includes most of PLCB4, and 10 other genes including JAG1, and consistent with JAG1 haploinsufficiency the patient was diagnosed with Alagille syndrome. No auricular abnormalities or micrognathia were reported. Patient 2009 is reported as having a duplication entirely within PLCB4 (encompassing exons 3–24; this duplication may give rise to a frameshifted protein), inherited from a normal parent, and a de novo deletion on chr17—no auricular or mandibular phenotypes are listed for the patient. Patient 263 696 is listed with a duplication encompassing a 3′ part of PLCB1 and a 5′ part of PLCB4 (these genes are contiguous, and arranged tail (PLCB1) >head (PLCB4), 5′ >3′), inherited from a normal parent, with no other CNVs. The phenotypes listed for this patient are cryptorchid testes and unilateral absent auditory canal and ossicles. This is intriguing given that auditory canal atresia and stenosis and conductive HL have been reported in ACS patients,2 ,3 ,5 ,15 and it would be worth investigating the possibility of an expressed PLCB4-PLCB1 fusion transcript in patient 263 696. We were unable to obtain further details for these latter two DECIPHER cases. Finally, several interstitial deletions of 20p12 have been reported in the literature, with probable heterozygous loss of PLCB4, but without an associated ACS phenotype (see Discussion).

Case 9 corresponds to the familial case M003 in Rieder et al,24 in which the PLCB4 mutation p.Arg621Cys was identified in the proband and his essentially asymptomatic father, while several relatives of the father were reported to display ACS-like features, without having been seen in the clinic. We present further details about the family that have come to light recently. The phenotype appears to have originated in the 13th and last child of the great-great-grandparents of the proband, suggestive of a neomutation on the paternal allele (figure 4A). We confirmed the presence of the p.Arg621Cys mutation in the paternal aunt (individual IV3). She presented, in contrast to her brother IV1 (figure 4C), ears typical of ACS (figure 4B), and had bilateral HL as a child but has a normal mandible. Typical ACS features can also be seen in individual III1 (figure 4D), who died at the age of 4 years from meningitis. The proband, in addition to a characteristic crease between the lobe and helix (figure 4E), displayed a postauricular tag (figure 4F) of a similar size and position to that of case 4 in the present report (see figure 3 in Gerkes et al9). A CT scan of the proband confirmed dysplasia of the mandibular ramus and condyle (figure 4G), and during surgery two hamartomatous pedicles were discovered in his throat; these may be similar in aetiology to previously reported soft tissue masses emanating from the posterior base of the mouth in ACS patients.5 ,24

Figure 4

Inheritance and auricular features of case 9, harbouring the PLCB4 mutation Arg621Cys. (A) case 9 pedigree. The PLCB4 mutant or wild-type allele is denoted by a minus or plus sign, respectively. A question mark indicates that no information was available. An asterisk indicates the proband. (B) individual IV3. (C) individual IV1. (D) individual III1. (E–G) individual V1, the proband. In (F) an arrow indicates the postauricular tag. (G) CT scan. This figure is only reproduced in colour in the online version.

Case 10 consists of an affected brother and sister born to healthy first-cousin parents. The brother presented with bifid uvula, laryngeal cleft, short velum, retrognathia, a typical QME on the right, a severely dysmorphic left ear and an aneurysm of the vein of Galen, while his sister displayed a left QME, over-folded helix on the right, glossoptosis and mandibular hypoplasia requiring distraction. Analysis of two microsatellites did not support homozygosity at the PLCB4 locus in the affected children. No mutations were identified in the entire coding region of PLCB4, GNAI3, the catalytic domain exons of PLCB3 (selected as a candidate given the branchial arch dysplasia in zebrafish harbouring plcb3 mutations29), or the coding region of GNAQ and GNA11, encoding G α proteins required for mandibular development in mice.35–38

Case 11 is a familial case of IQME, originating from Armenia. The mother presented with a significant notch in the border of the helix, at the lobe-helix junction, bilaterally. Her daughter displayed bilateral clefting between the lobe and helix. No overt signs of mandibular hypoplasia were evident. x-rays of the mandible of the daughter at 2 years of age did not reveal any obvious anomalies. No mutations were identified in the complete coding region of PLCB4, GNAI3, GNAQ, GNA11 or in the catalytic domain exons of PLCB3.

Case 12 consists of a mother, her daughter and her son, of African origin, all three presenting with IQME. The mother displayed a notch at the lobe-helix junction, and her son and daughter each had protruding helices and clefting, or constriction, between the lobe and helix. The daughter has been treated for acute lymphoblastic leukaemia. We were not able to assess mandibular x-rays in this family, but there were no external signs of micrognathia. We did not identify mutations in the complete coding region of PLCB4, GNAI3, GNAQ, GNA11 or in the catalytic domain exons of PLCB3.

We also sequenced the catalytic domain exons of PLCB4 and the coding region of GNAI3 in cases 1, 3, 4, 5 and 6 in McGowan et al,30 in six cases having been diagnosed as OAVS or GS, and in four cases with poorly defined ear dysplasias, including microtia or hypoplastic lobe, with or without mandibular dysplasia (cases 13–27 in table 1). No clearly pathogenic mutations were identified.

Mapping of mutated residues to protein structures

Previously, structural modelling of the four PLCB4 amino acid positions mutated in ACS patients indicated that they all occur within the catalytic domain (figure 5B and Rieder et al24). All four are predicted to form bonds with inositol triphosphate, the active-site calcium, or other amino acids involved in catalysis.24 Mapping the two novel mutated residues in this report, Glu358 and Asp360, to the PLCB3 crystal structure indicated that both are predicted to contact the active-site calcium (figure 5B). All six PLCB4 residues now known to be mutated in ACS exhibit a striking clustering in protein space (figure 5B). Mapping of Ser47, a novel mutation of which was identified in case 7 of this report, to the GNAI3 crystal structure, indicated that it makes contact with the active-site magnesium, which in turn coordinates the positioning of GTP (figure 5C and see ref. 39).


Following on from the initial description of PLCB4 and GNAI3 mutations in ACS, we tested both genes in several additional patients diagnosed with classic ACS, IQME, OAVS/GS and in others with less clearly defined auricular and mandibular dysplasias. Of 11 ACS/IQME patients tested, six harboured missense PLCB4 mutations, one had a homozygous deletion within PLCB4 predicted to result in complete loss of the protein, and one had a missense mutation in GNAI3. Combining the mutations reported here and in Rieder et al,24 12/15 solved cases (80%) have a PLCB4 lesion, and 3/15 (20%) have a GNAI3 mutation. Two PLCB4 amino acids emerge as hotspots: five out of the total of 11 PLCB4 point mutations now described occur at Arg621 (Arg621Cys x 2, Arg621His x 2, Arg621Leu) and two occur at Asp360 (Asp360Asn, Asp360Val). When mapped to the PLCB3 crystal structure, these two and all other mutated PLCB4 residues are predicted to form bonds important for catalysis in the active site.

We have elucidated the molecular basis of the first recessive case of ACS. Case 8 harboured a homozygous deletion of four exons within PLCB4, predicted to result in complete loss of the protein, with each mutant allele inherited from his consanguineous, unaffected parents, each of whom was heterozygous for the deletion. We note that patient 2 in Guion–Almeida et al5 was born to consanguineous parents and presented with ACS plus the atypical features of low birthweight, hypotonia, ptosis, down-slanting palpebral fissures, learning disability and delayed neuropsychomotor and language development. The possibility of homozygous loss-of-function alleles would be worth investigating in this patient. ACS was evoked in another patient born to consanguineous parents,40 but in our opinion, this diagnosis is unlikely to be correct. In addition to ACS, the patient carrying a homozygous deletion described here presented with central apnoea in repeated sleep studies. Although respiratory distress and presumed obstructive apnoea are commonly reported in ACS patients, central apnoea has not been described. After this finding, we analysed sleep studies performed in other ACS patients, and we found evidence for central events in some cases after distraction surgery, a procedure that should reduce obstructive apnoea events and unmask central events, if present. The potential role of PLCB4 in central control of respiration may be best addressed in Plcb4 homozygous null mice, under conditions that challenge respiration. Drawing on evidence from animal models harbouring mutations in genes of the endothelin signalling pathway, Rieder et al24 proposed that PLCB4 and GNAI3 may function downstream of endothelin signalling during branchial arch patterning. Interestingly, mutations in some endothelin signalling pathway components are associated with defects in respiratory control (reviewed in Gaultier et al41).

All previously described PLCB4 mutations were missense, clustered within the catalytic domain, and it was suggested that these mutations may result in dominant negative PLCB4 proteins.24 Support for this idea comes from evidence suggesting that the plcb3 missense mutations in the zebrafish schmerle mutant function as dominant negatives,29 and from a report of a PLCD4 splice variant that produces an isoform capable of dominantly interfering with other PLC family members.42 The phenotype, or lack thereof, of individuals with heterozygous PLCB4 deletions or frameshift or stop mutations was not described by Rieder et al.24 Individuals reported here, with heterozygous deletions of all or part of PLCB4, but without obvious features of ACS, provide strong evidence that the ACS-associated point mutations act as dominant negative rather than haploinsufficient alleles. In addition to the parents of case 8, we report two patients via DECIPHER, with deletions (likely heterozygous) that remove wholly or partly PLCB4, without leading to ACS. A series of five patients with overlapping microdeletions on chr20p12.3 associated with variably penetrant Wolff–Parkinson–White syndrome, dysmorphic features and neurocognitive delay has recently been reported, and although PLCB4 is contained within the two largest deletions (both apparently heterozygous), ears typical of ACS were not reported.43–45 JAG1, haploinsufficiency of which causes Alagille syndrome is 1.16 Mb centromeric to PLCB4. A number of deletions (likely heterozygous) in Alagille syndrome patients remove both JAG1 and PLCB4, but typical ACS phenotypes were not reported in these cases.46–48

A range of mostly neurological phenotypes have been described in Plcb4 knockout mice, including absence seizures, locomotor ataxia, defects in visual responses and processing, disruption of sleep sequence, increased anxiety and impaired fear extinction.49–56 However, craniofacial defects have not been reported in Plcb4 homozygous null mice. One hypothesis to explain the difference in phenotype between the missense mutations in humans and complete loss of function in mice would be that the mutant human PLCB4 protein dominantly interferes with multiple PLCB family members, and as noted above, this is supported by the evidence from the plcb3 mutations in zebrafish and from studies of a PLCD4 splice variant.29 ,42 However, the finding of classical ACS craniofacial features in a patient with homozygous loss of PLCB4 indicates that dominant interference with other PLCB family members is not necessary to produce the phenotype in humans. Indeed, the homozygous deletion case suggests that in the point mutation cases, dominant negative mutant PLCB4 only interferes with its wild-type PLCB4 counterpart in humans. Other possible reasons for the apparent discrepancy between human and mouse phenotypes could be that mice are better able to genetically compensate for the loss of Plcb4, or that subtle craniofacial defects do exist in the Plcb4 null mice, but are yet to be appreciated.

The only mutation previously reported in GNAI3 in ACS patients was Gly40Arg, occurring in two unrelated cases, with the mutation inherited from an affected parent in one case and from an unaffected parent in the other.24 Here we have identified a second GNAI3 mutation, Ser47Arg. Gly40 and Ser47 of GNAI3 corresponding to the first and last amino acids of the G1 box, one of five highly conserved motifs (G1–G5; figure 5A) involved in guanosine diphosphate (GDP)/guanosine triphosphate (GTP) binding by rat sarcoma (RAS) superfamily and G α proteins.57 All 16 human G α proteins conform to the G1 box consensus of GXXXSGKS. The hydroxyl of the final serine of the G1 box is involved in the coordination of magnesium during GTP binding by G α proteins.39 A mutation of the equivalent G1 box serine in HRAS (Ser17Asn) was shown to function as a dominant negative with growth-inhibitory properties,58 ,59 and subsequently a mutation at this position has been engineered in many RAS superfamily members and G α proteins, with consequent dominant negative effects (reviewed in Barren and Artemyev60). It was suggested by Rieder et al,24 that the GNAI3 Gly40Arg mutation in two ACS patients represents a gain-of-function mutation, however, we suggest that Ser47Arg is more likely a dominant negative, based on the in vitro data for other GTP-binding proteins mutated at this position. The GNAI3 mutation identified here was inherited from a father who was only mildly affected, consistent with the extreme phenotypic variability within families that occurs in other GNAI3 or PLCB4 mutation-positive cases. A similar phenomenon has been reported for other craniofacial disorders, for example, the non-penetrance for POLR1D mutations in some cases of Treacher Collins syndrome.61

Following exome sequencing of a series of ACS patients, Rieder et al24 were unable to identify PLCB4 or GNAI3 mutations in only one case (A001). However, a private missense mutation was identified in each of DOCK1 and DOCK6 in this patient. Given that DOCK genes code for guanine nucleotide exchange factors,62 and are therefore potential regulators of the G-protein-dependent pathway disrupted in ACS, it was proposed that these mutations may be responsible for ACS in patient A001, despite the absence of parental data. However, several arguments can be made against the putative involvement of DOCK1 and DOCK6 in ACS. Neither the DOCK1 nor DOCK6 mutation in patient A001 falls within the known domains of each protein (( and ( Recessive truncating mutations have been identified in DOCK6 as a cause of Adams–Oliver syndrome—the major features of this syndrome are digit truncations and scalp hypoplasia, but no craniofacial or ear phenotypes were reported in DOCK6-mutant patients, and no phenotypes were reported for their heterozygous parents.63 Murine Dock6 does not appear to be strongly expressed in the proximal mandible or external ear at several stages of embryonic development, as judged by the whole-mount in situ hybridisation images in Shaheen et al.63 DOCK1 falls within the smallest region of overlap of several 10q deletions, and although craniofacial dysmorphism is a component of the associated 10q deletion syndrome,64 to our knowledge, auricular defects of the ACS type have not been reported. For these reasons we did not sequence DOCK1 and DOCK6 in the PLCB4/GNAI3 mutation-negative patients reported here.

Whether IQME is genetically distinct from ACS, or whether it represents variable expression of the same disorder remains unknown. Indeed, we identified a PLCB4 mutation in a case previously reported as having IQME,18 however, in our opinion, the lower jaw is mildly dysplastic in two members of that family. Conversely, we sequenced PLCB4 and GNAI3 in two familial cases of IQME, without overt signs of mandibular hypoplasia, and we did not identify any mutations.

Within the ACS literature, postauricular tags have been reported in a small number of cases.2 ,3 ,9 ,33 We have shown here that the presence of a postauricular tag is a very strong indication of a PLCB4 mutation. All cases in this report with a postauricular tag have either a mutation at Arg621 or a homozygous deletion within PLCB4. Note that the position of the postauricular tag in the PLCB4 mutation-positive cases presented here is highly stereotypic, falling just above the lobe-helix junction. Although preauricular tags have been reported as a feature of several syndromes (perhaps most frequently in OAVS/GS), we are unaware of syndromes other than ACS with recurrence of postauricular tags. Although it has been claimed in the ACS literature that preauricular tags can occur as part of the phenotype, the evidence for this appears limited: preauricular tags were stated for individual IV-6 in Storm et al,3 but from the image in that article, we suggest that the preauricular tag may be a dysplastic tragus or antitragus; and for individual IV-3 in Masotti et al,2 although a classic postauricular tag is evident, the nature of the preauricular tag(s) is less clear. Along with the ear dysmorphology itself (see below), a post- versus a preauricular tag may be a useful feature in differentiating between ACS and other branchial arch anomalies.

It has been suggested that the auricular phenotype in ACS patients varies widely, and a recent report expanded the spectrum of ACS-associated anomalies to include ear phenotypes that might otherwise fall within OAVS/GS or other branchial arch syndromes.30 For some mutation-positive individuals in the familial cases of the present report, an auricular phenotype was non-penetrant, but when present, the auricular dysplasia in almost all cases consisted of an abnormal junction between the lobe and helix, ranging from a small indentation to a deep cleft. One exception to this was the father of case 5, who presented an over-folded helix as the only auricular dysmorphology, and an over-folded helix has been reported in other ACS cases.2 ,3 We sequenced all of GNAI3 and the catalytic domain of PLCB4 in five of the cases reported in McGowan et al,30 and in 10 patients with ear and craniofacial features consistent with OAVS/GS or with isolated pharyngeal arch anomalies, but no clearly pathogenic mutations were identified. None of the patients described in McGowan et al30 display the indentation or cleft between the helix and lobe that is characteristic of PLCB4/GNAI3 mutation-positive ACS cases. In some of the former cases, a cleft occurs between the tragus and antitragus. Based on the model whereby six auricular hillocks surrounding the first pharyngeal cleft give rise to specific portions of the external ear,65 it can be speculated that the boundary between the helix and lobe arises from the fusion of the fifth and sixth hillocks, while the tragus-anti-tragus boundary would arise from fusion across the first pharyngeal cleft. Our genotyping analysis supports the idea that the auricular phenotype in the patients reported in McGowan et al30 is caused by disruption of molecular and embryonic events distinct from those causing PLCB4/GNAI3-related ACS. We conclude that the auricular phenotype of PLCB4/GNAI3 mutation-positive ACS is in fact highly specific (notwithstanding the existence of both mild and severe forms within families), and distinguishable from other human ear dysplasias.


This work was supported by funding from the Ecole de l'Inserm Liliane Bettencourt (AV), the Jean Renny Endowment for Craniofacial Research (MLC), E-Rare CRANIRARE (SL) and the Fondation pour la Recherche Médicale (JA).


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  •  CTG and AV contributed equally

  • Contributors CTG, AV, SSP, AO, MS-B, EBB, MO, RP, BD and MS performed genetic analysis. SM, EG, AH, AA, MH-E, BS-R, AS, RM, FP, MRS, PA, DK, PP, J-PB, EST, SG-M, MB-G, PL, BAM, VA, FD, M-PV, CR-F, VC, SP, YM, SB, YMCH, AM, LJ, PK, AL, LBK, LB-V, LW, MLC, SL and JA diagnosed or treated patients. CTG, AV and JA wrote the manuscript.

  • Competing interests None.

  • Ethics approval CPP Ile-de-France II.

  • Patient consent Obtained.

  • Provenance and peer review Not commissioned; externally peer reviewed.

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