A Human Homeotic Transformation Resulting from Mutations in PLCB4 and GNAI3 Causes Auriculocondylar Syndrome
2012; Elsevier BV; Volume: 90; Issue: 5 Linguagem: Inglês
10.1016/j.ajhg.2012.04.002
ISSN1537-6605
AutoresMark J. Rieder, Glenn E. Green, Sarah S. Park, Brendan D. Stamper, Christopher T. Gordon, Jason M. Johnson, Christopher Cunniff, Joshua D. Smith, Sarah B. Emery, Stanislas Lyonnet, Jeanne Amiel, Muriel Holder, Andrew A. Heggie, Michael J. Bamshad, Deborah A. Nickerson, Timothy C. Cox, Anne Hing, Jeremy A. Horst, Michael L. Cunningham,
Tópico(s)dental development and anomalies
ResumoAuriculocondylar syndrome (ACS) is a rare, autosomal-dominant craniofacial malformation syndrome characterized by variable micrognathia, temporomandibular joint ankylosis, cleft palate, and a characteristic “question-mark” ear malformation. Careful phenotypic characterization of severely affected probands in our cohort suggested the presence of a mandibular patterning defect resulting in a maxillary phenotype (i.e., homeotic transformation). We used exome sequencing of five probands and identified two novel (exclusive to the patient and/or family studied) missense mutations in PLCB4 and a shared mutation in GNAI3 in two unrelated probands. In confirmatory studies, three additional novel PLCB4 mutations were found in multigenerational ACS pedigrees. All mutations were confirmed by Sanger sequencing, were not present in more than 10,000 control chromosomes, and resulted in amino-acid substitutions located in highly conserved protein domains. Additionally, protein-structure modeling demonstrated that all ACS substitutions disrupt the catalytic sites of PLCB4 and GNAI3. We suggest that PLCB4 and GNAI3 are core signaling molecules of the endothelin-1-distal-less homeobox 5 and 6 (EDN1-DLX5/DLX6) pathway. Functional studies demonstrated a significant reduction in downstream DLX5 and DLX6 expression in ACS cases in assays using cultured osteoblasts from probands and controls. These results support the role of the previously implicated EDN1-DLX5/6 pathway in regulating mandibular specification in other species, which, when disrupted, results in a maxillary phenotype. This work defines the molecular basis of ACS as a homeotic transformation (mandible to maxilla) in humans. Auriculocondylar syndrome (ACS) is a rare, autosomal-dominant craniofacial malformation syndrome characterized by variable micrognathia, temporomandibular joint ankylosis, cleft palate, and a characteristic “question-mark” ear malformation. Careful phenotypic characterization of severely affected probands in our cohort suggested the presence of a mandibular patterning defect resulting in a maxillary phenotype (i.e., homeotic transformation). We used exome sequencing of five probands and identified two novel (exclusive to the patient and/or family studied) missense mutations in PLCB4 and a shared mutation in GNAI3 in two unrelated probands. In confirmatory studies, three additional novel PLCB4 mutations were found in multigenerational ACS pedigrees. All mutations were confirmed by Sanger sequencing, were not present in more than 10,000 control chromosomes, and resulted in amino-acid substitutions located in highly conserved protein domains. Additionally, protein-structure modeling demonstrated that all ACS substitutions disrupt the catalytic sites of PLCB4 and GNAI3. We suggest that PLCB4 and GNAI3 are core signaling molecules of the endothelin-1-distal-less homeobox 5 and 6 (EDN1-DLX5/DLX6) pathway. Functional studies demonstrated a significant reduction in downstream DLX5 and DLX6 expression in ACS cases in assays using cultured osteoblasts from probands and controls. These results support the role of the previously implicated EDN1-DLX5/6 pathway in regulating mandibular specification in other species, which, when disrupted, results in a maxillary phenotype. This work defines the molecular basis of ACS as a homeotic transformation (mandible to maxilla) in humans. Auriculocondylar syndrome (ACS; MIM 602483; also known as “question-mark ear syndrome” or “dysgnathia complex”) is an autosomal-dominant craniofacial malformation syndrome characterized by highly variable mandibular anomalies ranging from mild to severe micrognathia, often with temporomandibular joint (TMJ) ankylosis, cleft palate, and a distinctive ear malformation that consists of separation of the lobule from the external ear, giving the appearance of a question mark (Figure 1). Other frequently described features include prominent cheeks, cupped and posteriorly rotated ears, preauricular tags, and microstomia. ACS was first described in a mother and her two affected children just over thirty years ago,1Uuspää V. Combined bilateral external ear deformity and hypoplastic mandible. Case report.Scand. J. Plast. Reconstr. Surg. 1978; 12: 165-167Crossref PubMed Scopus (24) Google Scholar and nine well-characterized families with multiple affected individuals2Jampol M. Repetto G. Keith D.A. Curtin H. Remensynder J. Holmes L.B. New syndrome? Prominent, constricted ears with malformed condyle of the mandible.Am. J. Med. Genet. 1998; 75: 449-452Crossref PubMed Scopus (25) Google Scholar, 3Erlich M.S. Cunningham M.L. Hudgins L. Transmission of the dysgnathia complex from mother to daughter.Am. J. Med. Genet. 2000; 95: 269-274Crossref PubMed Scopus (47) Google Scholar, 4Guion-Almeida M.L. Zechi-Ceide R.M. Vendramini S. Kokitsu-Nakata N.M. Auriculo-condylar syndrome: additional patients.Am. J. Med. Genet. 2002; 112: 209-214Crossref PubMed Scopus (28) Google Scholar, 5Storm A.L. Johnson J.M. Lammer E. Green G.E. Cunniff C. Auriculo-condylar syndrome is associated with highly variable ear and mandibular defects in multiple kindreds.Am. J. Med. Genet. A. 2005; 138A: 141-145Crossref PubMed Scopus (25) Google Scholar, 6Masotti C. Oliveira K.G. Poerner F. Splendore A. Souza J. Freitas Rda.S. Zechi-Ceide R. Guion-Almeida M.L. Passos-Bueno M.R. Auriculo-condylar syndrome: mapping of a first locus and evidence for genetic heterogeneity.Eur. J. Hum. Genet. 2008; 16: 145-152Crossref PubMed Scopus (24) Google Scholar, 7Ozturk S. Sengezer M. Isik S. Gul D. Zor F. The correction of auricular and mandibular deformities in auriculo-condylar syndrome.J. Craniofac. Surg. 2005; 16: 489-492Crossref PubMed Scopus (12) Google Scholar, 8Shkalim V. Eliaz N. Linder N. Merlob P. Basel-Vanagaite L. Autosomal dominant isolated question mark ear.Am. J. Med. Genet. A. 2008; 146A: 2280-2283Crossref PubMed Scopus (9) Google Scholar (and at least one linkage peak mapping to 1p21-1q236Masotti C. Oliveira K.G. Poerner F. Splendore A. Souza J. Freitas Rda.S. Zechi-Ceide R. Guion-Almeida M.L. Passos-Bueno M.R. Auriculo-condylar syndrome: mapping of a first locus and evidence for genetic heterogeneity.Eur. J. Hum. Genet. 2008; 16: 145-152Crossref PubMed Scopus (24) Google Scholar, 9Kokitsu-Nakata N.M. Zechi-Ceide R.M. Vendramini-Pittoli S. Romanelli Tavares V.L. Passos-Bueno M.R. Guion-Almeida M.L. Auriculo-condylar syndrome. Confronting a diagnostic challenge.Am. J. Med. Genet. A. 2011; (in press. Published online November 21, 2011)https://doi.org/10.1002/ajmg.a.34337Crossref PubMed Scopus (13) Google Scholar) as well as several simplex cases4Guion-Almeida M.L. Zechi-Ceide R.M. Vendramini S. Kokitsu-Nakata N.M. Auriculo-condylar syndrome: additional patients.Am. J. Med. Genet. 2002; 112: 209-214Crossref PubMed Scopus (28) Google Scholar, 10Guion-Almeida M.L. Kokitsu-Nakata N.M. Zechi-Ceide R.M. Vendramini S. Auriculo-condylar syndrome: further evidence for a new disorder.Am. J. Med. Genet. 1999; 86: 130-133Crossref PubMed Scopus (16) Google Scholar, 11Priolo M. Lerone M. Rosaia L. Calcagno E.P. Sadeghi A.K. Ghezzi F. Ravazzolo R. Silengo M. Question mark ears, temporo-mandibular joint malformation and hypotonia: auriculo-condylar syndrome or a distinct entity?.Clin. Dysmorphol. 2000; 9: 277-280Crossref PubMed Scopus (24) Google Scholar, 12Gerkes E.H. van Ravenswaaij C.M. van Essen A.J. Question mark ears and post-auricular tags.Eur. J. Med. Genet. 2008; 51: 264-267Crossref PubMed Scopus (8) Google Scholar have been reported to date. Our index case (S011) and her affected mother (S012P; kindred D, individuals I-1 and II-1, respectively, in Figure S1, available online) had severe mandibular abnormalities:3Erlich M.S. Cunningham M.L. Hudgins L. Transmission of the dysgnathia complex from mother to daughter.Am. J. Med. Genet. 2000; 95: 269-274Crossref PubMed Scopus (47) Google Scholar congenital mandibular ankylosis with lateral fusion of the mandible to the temporozygomatic suture and medial fusion to the skull base (medial and lateral pterygoid plates) (Figure 2), resulting in airway obstruction requiring tracheostomy. Intraoperative evaluation of S011 was notable for the presence of severe microglossia, elongated soft-tissue masses attached at the posterior floor of the mouth, and excessive soft tissue protruding from the medial alveolus of the lower jaw, giving the appearance of a “mandibular palate” (Figure 2). We identified four additional kindreds with dysgnathia complex (i.e., ACS),3Erlich M.S. Cunningham M.L. Hudgins L. Transmission of the dysgnathia complex from mother to daughter.Am. J. Med. Genet. 2000; 95: 269-274Crossref PubMed Scopus (47) Google Scholar as well as two simplex case child-parent trios (S001 and S008 [II-1 individuals in kindreds B and C, respectively, in Figure S1]), a family with two affected siblings (S004 [proband] and S005 [kindred A, individuals II-1 and II-2 in Figure S1]) whose father had mild mandibular hypoplasia, and an additional singleton proband (A001; Figure S1). We obtained approval for this study from our institutional review board (Seattle Children's Hospital), written consent was obtained for all participants, and consent for photography was obtained from all individuals whose images are included in this manuscript. In each case, mandibular ankylosis was progressive, of variable severity, and characterized by inconsistent fusion to the medial and lateral pterygoid plates. All cases demonstrated a similar phenotype, consisting of a lateral mandibular bony prominence with or without TMJ ankylosis, and had features consistent with classic ACS. The anatomic features of these cases led us to hypothesize that the malformations observed in individuals with ACS were due to a homeotic transformation, with the mandible assuming a maxillary phenotype (Figures 2E–2G). Other reports of mandibular patterning mutations in mice and zebrafish have implicated the endothelin-1 (EDN1) receptor A (EDNRA) and its downstream targets, distal-less homeobox 5 and 6 (DLX5 and DLX6).13Vieux-Rochas M. Mantero S. Heude E. Barbieri O. Astigiano S. Couly G. Kurihara H. Levi G. Merlo G.R. Spatio-temporal dynamics of gene expression of the Edn1-Dlx5/6 pathway during development of the lower jaw.Genesis. 2010; 48: 262-373Crossref PubMed Scopus (22) Google Scholar, 14Ivey K. Tyson B. Ukidwe P. McFadden D.G. Levi G. Olson E.N. Srivastava D. Wilkie T.M. Galphaq and Galpha11 proteins mediate endothelin-1 signaling in neural crest-derived pharyngeal arch mesenchyme.Dev. Biol. 2003; 255: 230-237Crossref PubMed Scopus (48) Google Scholar, 15Depew M.J. Lufkin T. Rubenstein J.L. Specification of jaw subdivisions by Dlx genes.Science. 2002; 298: 381-385Crossref PubMed Scopus (370) Google Scholar, 16Beverdam A. Merlo G.R. Paleari L. Mantero S. Genova F. Barbieri O. Janvier P. Levi G. Jaw transformation with gain of symmetry after Dlx5/Dlx6 inactivation: mirror of the past?.Genesis. 2002; 34: 221-227Crossref PubMed Scopus (150) Google Scholar Sanger sequencing of endothelin-1 pathway candidates EDNRA, DLX5, and DLX6 in our probands was negative. We performed exome sequencing on all five probands and parents (excluding A001 [no parental DNA available] and S012-P [inadequate sample]) to identify gene-based coding variants present in individuals with ACS. Genomic libraries were prepared and underwent exome capture with the use of a 28 Mb target derived from the consensus coding sequence (CCDS) database (version 20080902)17Ng S.B. Buckingham K.J. Lee C. Bigham A.W. Tabor H.K. Dent K.M. Huff C.D. Shannon P.T. Jabs E.W. Nickerson D.A. et al.Exome sequencing identifies the cause of a mendelian disorder.Nat. Genet. 2010; 42: 30-35Crossref PubMed Scopus (1484) Google Scholar or the ∼32 Mb target from Roche Nimblegen SeqCap EZ (version 1.0). Exome-enriched libraries were sequenced on Illumina GAIIx or HiSeq 2000 platforms with paired-end 50 or 76 base reads. Each individual generated ∼75 million unique reads mapping to the exome target and nearby flanking regions. Over 93% of the exome positions had a coverage depth > 8×, and the average overall coverage depth was 103×. Data from each individual were processed from real-time base calls and aligned to a human reference (hg19) through the use of the Burrows-Wheeler Aligner.18Li H. Durbin R. Fast and accurate short read alignment with Burrows-Wheeler transform.Bioinformatics. 2009; 25: 1754-1760Crossref PubMed Scopus (26642) Google Scholar Data was processed with the use of the Genome Analysis Toolkit19DePristo M.A. Banks E. Poplin R. Garimella K.V. Maguire J.R. Hartl C. Philippakis A.A. del Angel G. Rivas M.A. Hanna M. et al.A framework for variation discovery and genotyping using next-generation DNA sequencing data.Nat. Genet. 2011; 43: 491-498Crossref PubMed Scopus (7098) Google Scholar (GATK reference version 1.0.2905), and variant detection and genotyping were performed with the use of the Unified Genotyper (GATK) tool. We flagged variant sites by using the filtration walker (GATK) to mark sites that were of lower quality and more likely to be false positives. Each individual generated an average of ∼22,000 total variants, with a final-pass filter set of ∼18,900 variants annotated for variant function (i.e., missense, synonymous, splicing, insertion or deletion [in/del], etc.) (Genome Variation Server). Given the consistency among the phenotypic features, we first analyzed exome data from all five probands to identify any genes with novel variants shared among them. Proband variant data (i.e., missense, in/dels, and splice acceptor and donor sites) were initially filtered against a smaller database of ∼1,200 control exomes drawn from a subset of individuals from the National Heart, Lung, and Blood Institute (NHLBI) Exome Sequencing Project (ESP) to identify novel variants, those passing GATK filtering, and those evident at a robust coverage level (>20x). Second, we performed discrete filtering to identify any gene in which all probands shared novel missense, insertion/deletion, nonsense, or splice variants consistent with either an autosomal-dominant or -recessive model. Under these models and criteria, no genes were detected in common between all probands. We expanded our search for de novo mutations in probands (S001, S004, and S008), using data from parental individuals along with stringent parameters for GATK pass filters and a sequence depth of >20× in both parents and probands. This identified six putative genes (ASPSCR1, C2orf16, HERC1, PAIP1, PLCB4, and TCHP) within these three individuals. The PLCB4 (MIM 600810) variant c.1868A>G (NM_000933.3; p.Tyr623Cys) had the highest conservation Genomic Evolutionary Rate Profiling (GERP)20Cooper G.M. Stone E.A. Asimenos G. Green E.D. Batzoglou S. Sidow A. NISC Comparative Sequencing ProgramDistribution and intensity of constraint in mammalian genomic sequence.Genome Res. 2005; 15: 901-913Crossref PubMed Scopus (957) Google Scholar, 21Cooper G.M. Goode D.L. Ng S.B. Sidow A. Bamshad M.J. Shendure J. Nickerson D.A. Single-nucleotide evolutionary constraint scores highlight disease-causing mutations.Nat. Methods. 2010; 7: 250-251Crossref PubMed Scopus (137) Google Scholar score (6.17) and was considered the top candidate on the basis of the core signaling role of its ortholog plcb3 in mandibular patterning of zebrafish.22Walker M.B. Miller C.T. Swartz M.E. Eberhart J.K. Kimmel C.B. phospholipase C, beta 3 is required for Endothelin1 regulation of pharyngeal arch patterning in zebrafish.Dev. Biol. 2007; 304: 194-207Crossref PubMed Scopus (56) Google Scholar, 23Walker M.B. Miller C.T. Coffin Talbot J. Stock D.W. Kimmel C.B. Zebrafish furin mutants reveal intricacies in regulating Endothelin1 signaling in craniofacial patterning.Dev. Biol. 2006; 295: 194-205Crossref PubMed Scopus (87) Google Scholar Sanger sequencing of parents S002-P and S003-P (kindred B, individuals I-1 and I-2 in Figure S1) and the proband (S001) confirmed it as a de novo mutation (Table 1). On the basis of this finding, we searched the remaining proband exome data for additional PLCB4 variants and identified, in siblings S004 and S005, a second missense mutation at c.986A>C, which changed the amino acid at position 329 from asparagine to serine (p.Asn329Ser; Table 1). In this kindred, the mutation was transmitted from the mildly affected father (S006-P [kindred A, individual I-1 in Figure S1]), a transmission which supports the variable expressivity of ACS.Table 1PLCB4 and GNAI3 Mutations in Probands with Auriculocondylar SyndromeIndividual/ PedigreeGeneAmino AcidNucleotideGERPGSControl FrequencyInheritanceS001PLCB4Tyr623Cysc.1868A>G6.171940/10758de novoS004aProband has a similarly affected sibling and mildly affected father with the same mutation.37PLCB4Asn329Serc.986A>C5.93460/10758transmitted (IPbIP, incomplete penetrance.)M001PLCB4Arg621Hisc.1862G>A6.17290/10758transmittedM002PLCB4Asn650Hisc.1948A>C5.92680/10758transmittedM003PLCB4Arg621Cysc.1861C>T6.171800/10758transmitted (IPbIP, incomplete penetrance.)S008, S011GNAI3Gly40Argc.118G>C5.821250/10758transmittedExome sequencing and Sanger confirmation identified five missense mutations that lead to rare substitutions in the catalytic domain of PLCB4 in five families and a single GNAI3 catalytic domain substitution in two unrelated families. These mutations are in highly conserved domains as indicated by GERP score and Grantham score (GS), and they are not found in a control set of human exomes from the NHLBI ESP data release ESP5400, accessed on December 10, 2011. M001 and M002 are families 1 and 2, respectively, as described in Storm et al.5Storm A.L. Johnson J.M. Lammer E. Green G.E. Cunniff C. Auriculo-condylar syndrome is associated with highly variable ear and mandibular defects in multiple kindreds.Am. J. Med. Genet. A. 2005; 138A: 141-145Crossref PubMed Scopus (25) Google Scholara Proband has a similarly affected sibling and mildly affected father with the same mutation.37Kano M. Hashimoto K. Watanabe M. Kurihara H. Offermanns S. Jiang H. Wu Y. Jun K. Shin H.S. Inoue Y. et al.Phospholipase cbeta4 is specifically involved in climbing fiber synapse elimination in the developing cerebellum.Proc. Natl. Acad. Sci. USA. 1998; 95: 15724-15729Crossref PubMed Scopus (163) Google Scholarb IP, incomplete penetrance. Open table in a new tab Exome sequencing and Sanger confirmation identified five missense mutations that lead to rare substitutions in the catalytic domain of PLCB4 in five families and a single GNAI3 catalytic domain substitution in two unrelated families. These mutations are in highly conserved domains as indicated by GERP score and Grantham score (GS), and they are not found in a control set of human exomes from the NHLBI ESP data release ESP5400, accessed on December 10, 2011. M001 and M002 are families 1 and 2, respectively, as described in Storm et al.5Storm A.L. Johnson J.M. Lammer E. Green G.E. Cunniff C. Auriculo-condylar syndrome is associated with highly variable ear and mandibular defects in multiple kindreds.Am. J. Med. Genet. A. 2005; 138A: 141-145Crossref PubMed Scopus (25) Google Scholar The discovery of putative coding-sequence, disease-causing PLCB4 mutations in isolated ACS kindreds led us to use Sanger sequencing to screen the gene coding regions containing the conserved catalytic site (exons 11–26) in two additional multigeneration ACS pedigrees5Storm A.L. Johnson J.M. Lammer E. Green G.E. Cunniff C. Auriculo-condylar syndrome is associated with highly variable ear and mandibular defects in multiple kindreds.Am. J. Med. Genet. A. 2005; 138A: 141-145Crossref PubMed Scopus (25) Google Scholar (family 1 [M001] and family 2 [M002] in Figure S2). We identified two additional novel PLCB4 missense mutations at c.1862G>A (M001) and c.1948A>C (M002) (with resultant protein changes, p.Asn650His and p.Arg621His, respectively) that segregated in affected individuals (Table 1 and Figure S2). We have also identified a family with an ACS proband (family 3 [M003], individual IV-1 in Figure S2; with micrognathia, cleft palate, glossoptosis, and a constriction between the helix and the lobule of the left ear) and his “unaffected” father (individual III-1) who presented with asymmetric ear lobes. The family history is significant for four paternal relatives with ear malformations with or without micrognathia (Table 1; family 3 in Figure S2). Sanger sequencing confirmed a PLCB4 missense mutation at c.1862G>A (p.Arg621Cys) in the proband and his unaffected father, the same amino-acid position (621) as noted in family M001 but a different amino-acid change. The phenotypic variability of ACS in these families was confirmed by the variable expressivity and apparent incomplete penetrance associated with the father (S006-P) of a proband with a PLCB4 mutation.5Storm A.L. Johnson J.M. Lammer E. Green G.E. Cunniff C. Auriculo-condylar syndrome is associated with highly variable ear and mandibular defects in multiple kindreds.Am. J. Med. Genet. A. 2005; 138A: 141-145Crossref PubMed Scopus (25) Google Scholar None of the PLCB4 mutations we discovered had been identified previously in a large set of control exomes containing 10,758 chromosomes (Table 1). Each of the mutations led to substitutions at highly conserved nucleotide positions (those with a GERP score > 5.8 make up the top 0.006% of coding nucleotides21Cooper G.M. Goode D.L. Ng S.B. Sidow A. Bamshad M.J. Shendure J. Nickerson D.A. Single-nucleotide evolutionary constraint scores highlight disease-causing mutations.Nat. Methods. 2010; 7: 250-251Crossref PubMed Scopus (137) Google Scholar) and evolutionary invariant amino-acid positions (Figure 3). Discrete filtering of exome data from the PLCB4-mutation-negative probands (S008, S011, and A001) revealed a Sanger-confirmed single-missense mutation in an inhibitory G protein (GNAI3; MIM 139370) in exon 1 (c.118G>C [NM_006496.2; p.Gly40Arg]) shared by individuals S008 and S011 (Table 1). In both kindreds, the p.Gly40Arg variant was inherited; in one case (S011) it was inherited from an affected mother (S012-P), and in the other (S008), from an unaffected father (S009-P), documenting incomplete penetrance. Using the exome data, we confirmed that these individuals did not share a haplotype region encompassing this mutation, and all other measures of global genomic relatedness, determined by analyzing all exome variants or specific high-frequency variants, were consistent with unrelatedness. These data suggest the possibility of a recurrent mutation at this position. The p.Gly40Arg substitution also was not observed in 10,758 control chromosomes, and it occurred at a highly conserved amino-acid residue (and the Grantham score [125] suggested a moderately radical amino-acid replacement24Li W.H. Wu C.I. Luo C.C. Nonrandomness of point mutation as reflected in nucleotide substitutions in pseudogenes and its evolutionary implications.J. Mol. Evol. 1984; 21: 58-71Crossref PubMed Scopus (273) Google Scholar) within the guanosine diphosphate (GDP)-binding catalytic domain of this G protein (Table 1; Figure 3). Furthermore, GNAI3 maps in the vicinity of the region on chromosome 1 previously linked with the ACS phenotype.6Masotti C. Oliveira K.G. Poerner F. Splendore A. Souza J. Freitas Rda.S. Zechi-Ceide R. Guion-Almeida M.L. Passos-Bueno M.R. Auriculo-condylar syndrome: mapping of a first locus and evidence for genetic heterogeneity.Eur. J. Hum. Genet. 2008; 16: 145-152Crossref PubMed Scopus (24) Google Scholar Given this, we considered the GNAI3 p.Gly40Arg change to be an ACS candidate because of the known role of phospholipase C (PLC) in G protein-coupled receptor signaling, a core component of the endothelin-DLX pathway14Ivey K. Tyson B. Ukidwe P. McFadden D.G. Levi G. Olson E.N. Srivastava D. Wilkie T.M. Galphaq and Galpha11 proteins mediate endothelin-1 signaling in neural crest-derived pharyngeal arch mesenchyme.Dev. Biol. 2003; 255: 230-237Crossref PubMed Scopus (48) Google Scholar (reviewed in Clauthia et al.25Clouthier D.E. Garcia E. Schilling T.F. Regulation of facial morphogenesis by endothelin signaling: insights from mice and fish.Am. J. Med. Genet. A. 2010; 152A: 2962-2973Crossref PubMed Scopus (70) Google Scholar). To further evaluate the significance of variants in PLCB4 (p.Asn329Ser, p.Arg621His, p.Arg621Cys, p.Tyr623Cys, p.Asn650His) and GNAI3 (p.Gly40Arg), we performed detailed structural protein modeling of specific substitutions found in ACS probands. All missense variants observed in cases, controls, and accessible databases were mapped to PLCB4 and GNAI3 protein structures and visualized with UCSF Chimera.26Pettersen E.F. Goddard T.D. Huang C.C. Couch G.S. Greenblatt D.M. Meng E.C. Ferrin T.E. UCSF Chimera—a visualization system for exploratory research and analysis.J. Comput. Chem. 2004; 25: 1605-1612Crossref PubMed Scopus (27946) Google Scholar None of the variants identified in controls occurred in the catalytic domains of these highly conserved proteins. In PLCB4, modeling each mutated side chain resulted in minimal overall structural impact27Ngan S.C. Hung L.H. Liu T. Samudrala R. Scoring functions for de novo protein structure prediction revisited.Methods Mol. Biol. 2008; 413: 243-281PubMed Google Scholar but removed hydrogen and ionic bonds essential to the catalysis of phosphatidylinositol 4,5-bisphosphate into inositol 1,4,5-trisphosphate and diacylglycerol (Figures 3C and 3D). Thus, these ACS-associated PLCB4 substitutions are localized within the PLCB4 catalytic substrate binding domain (Figure 3D) and would be expected to directly affect function. The GNAI3 p.Gly40Arg variant side chain was modeled with each of the three currently available GNAI3 structures.28Soundararajan M. Willard F.S. Kimple A.J. Turnbull A.P. Ball L.J. Schoch G.A. Gileadi C. Fedorov O.Y. Dowler E.F. Higman V.A. et al.Structural diversity in the RGS domain and its interaction with heterotrimeric G protein alpha-subunits.Proc. Natl. Acad. Sci. USA. 2008; 105: 6457-6462Crossref PubMed Scopus (139) Google Scholar, 29Kimple A.J. Soundararajan M. Hutsell S.Q. Roos A.K. Urban D.J. Setola V. Temple B.R. Roth B.L. Knapp S. Willard F.S. Siderovski D.P. Structural determinants of G-protein alpha subunit selectivity by regulator of G-protein signaling 2 (RGS2).J. Biol. Chem. 2009; 284: 19402-19411Crossref PubMed Scopus (56) Google Scholar Effects of the substitution on GNAI3 stability were measured with multiple knowledge-based scoring functions.27Ngan S.C. Hung L.H. Liu T. Samudrala R. Scoring functions for de novo protein structure prediction revisited.Methods Mol. Biol. 2008; 413: 243-281PubMed Google Scholar Minimal to positive influence on stability suggested further study and led us to examine why this large arginine substitution was tolerated in the conserved core of the G-alpha protein.27Ngan S.C. Hung L.H. Liu T. Samudrala R. Scoring functions for de novo protein structure prediction revisited.Methods Mol. Biol. 2008; 413: 243-281PubMed Google Scholar A detailed assessment30Dundas J. Ouyang Z. Tseng J. Binkowski A. Turpaz Y. Liang J. CASTp: computed atlas of surface topography of proteins with structural and topographical mapping of functionally annotated residues.Nucleic Acids Res. 2006; 34: W116-W118Crossref PubMed Scopus (1410) Google Scholar revealed a cavity large enough for an organic cationic metabolite in each of the three crystal structures (Figures 3E and 3F, semi-transparent green surface) and large enough to hold and electrostatically balance the guanidinium group. Our protein modeling suggested the presence of hydrogen bonds between the Arg40 side-chain nitrogens and (1) the P loop Gly42 backbone carbonyl, (2) the Asp229 side chain, and (3) the Ser246 side chain (Figure 3F, pink lines). Modeling the p.Gly40Arg variant suggests stabilization of GNAI3 in the active conformation (switch III).25Clouthier D.E. Garcia E. Schilling T.F. Regulation of facial morphogenesis by endothelin signaling: insights from mice and fish.Am. J. Med. Genet. A. 2010; 152A: 2962-2973Crossref PubMed Scopus (70) Google Scholar Examination of potential interactions with the downstream signaling partner RapGAPII through analysis of the common structural fold between Rap and GNAI331Scrima A. Thomas C. Deaconescu D. Wittinghofer A. The Rap-RapGAP complex: GTP hydrolysis without catalytic glutamine and arginine residues.EMBO J. 2008; 27: 1145-1153Crossref PubMed Scopus (91) Google Scholar revealed close recognition of the constitutively activated switch III. Thus, modeling the GNAI3 p.Gly40Arg substitution suggests a gain of function, with stabilization of the active conformation recognized by RapGapII triggering inhibition of the mitogen-activated protein kinase (MAPK) pathway activation through Rap. 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