Asymmetric Lower-Limb Malformations in Individuals with Homeobox PITX1 Gene Mutation
2008; Elsevier BV; Volume: 83; Issue: 5 Linguagem: Inglês
10.1016/j.ajhg.2008.10.004
ISSN1537-6605
AutoresChristina A. Gurnett, Farhang Alaee, Lisa M. Kruse, David M. Desruisseau, Jacqueline T. Hecht, Carol A. Wise, A. Bowcock, Matthew B. Dobbs,
Tópico(s)Neurogenetic and Muscular Disorders Research
ResumoClubfoot is one of the most common severe musculoskeletal birth defects, with a worldwide incidence of 1 in 1000 live births. In the present study, we describe a five-generation family with asymmetric right-sided predominant idiopathic clubfoot segregating as an autosomal-dominant condition with incomplete penetrance. Other lower-limb malformations, including patellar hypoplasia, oblique talus, tibial hemimelia, developmental hip dysplasia, and preaxial polydactyly, were also present in some family members. Genome-wide linkage analysis with Affymetrix GeneChip Mapping 10K mapping data from 13 members of this family revealed a multipoint LODmax of 3.31 on chromosome 5q31. A single missense mutation (c.388G→A) was identified in PITX1, a bicoid-related homeodomain transcription factor critical for hindlimb development, and segregated with disease in this family. The PITX1 E130K mutation is located in the highly conserved homeodomain and reduces the ability of PITX1 to transactivate a luciferase reporter. The PITX1 E130K mutation also suppresses wild-type PITX1 activity in a dose-dependent manner, suggesting dominant-negative effects on transcription. The propensity for right-sided involvement in tibial hemimelia and clubfoot suggests that PITX1, or pathways involving PITX1, may be involved in their etiology. Implication of a gene involved in early limb development in clubfoot pathogenesis also suggests additional pathways for future investigations of idiopathic clubfoot etiology in humans. Clubfoot is one of the most common severe musculoskeletal birth defects, with a worldwide incidence of 1 in 1000 live births. In the present study, we describe a five-generation family with asymmetric right-sided predominant idiopathic clubfoot segregating as an autosomal-dominant condition with incomplete penetrance. Other lower-limb malformations, including patellar hypoplasia, oblique talus, tibial hemimelia, developmental hip dysplasia, and preaxial polydactyly, were also present in some family members. Genome-wide linkage analysis with Affymetrix GeneChip Mapping 10K mapping data from 13 members of this family revealed a multipoint LODmax of 3.31 on chromosome 5q31. A single missense mutation (c.388G→A) was identified in PITX1, a bicoid-related homeodomain transcription factor critical for hindlimb development, and segregated with disease in this family. The PITX1 E130K mutation is located in the highly conserved homeodomain and reduces the ability of PITX1 to transactivate a luciferase reporter. The PITX1 E130K mutation also suppresses wild-type PITX1 activity in a dose-dependent manner, suggesting dominant-negative effects on transcription. The propensity for right-sided involvement in tibial hemimelia and clubfoot suggests that PITX1, or pathways involving PITX1, may be involved in their etiology. Implication of a gene involved in early limb development in clubfoot pathogenesis also suggests additional pathways for future investigations of idiopathic clubfoot etiology in humans. Several congenital limb malformations exclusively affect the lower limb, including developmental hip dysplasia, proximal focal femoral deficiency, tibial and fibular hemimelia, and clubfoot (talipes equinovarus). Clubfoot is one of the most common serious congenital musculoskeletal anomalies with a worldwide incidence of 1 in 1000 live births.1Wynne-Davies R. Genetic and environmental factors in the etiology of talipes equinovarus.Clin. Orthop. Relat. Res. 1972; 84: 9-13Crossref PubMed Scopus (126) Google Scholar Approximately 80% of clubfeet occur as isolated birth defects and are considered idiopathic.2Wynne-Davies R. Family studies and the cause Of congenital club foot. Talipes equinovarus, talipes calcaneo-valgus and metatarsus varus.J. Bone Joint Surg. Br. 1964; 46: 445-463PubMed Google Scholar Genetic factors play a role in the etiology of clubfoot, given that nearly 25% of all cases are familial.3Lochmiller C. Johnston D. Scott A. Risman M. Hecht J.T. Genetic epidemiology study of idiopathic talipes equinovarus.Am. J. Med. Genet. 1998; 79: 90-96Crossref PubMed Scopus (135) Google Scholar Additional evidence for a genetic etiology is provided by differences in clubfoot prevalence across ethnic populations with the lowest prevalence in Chinese (0.39 cases per 1,000 live births) and the highest in Hawaiians and Maoris (7 per 1,000).4Chung C.S. Nemechek R.W. Larsen I.J. Ching G.H. Genetic and epidemiological studies of clubfoot in Hawaii. General and medical considerations.Hum. Hered. 1969; 19: 321-342Crossref PubMed Scopus (66) Google Scholar, 5Beals R.K. Club foot in the Maori: A genetic study of 50 kindreds.N. Z. Med. J. 1978; 88: 144-146PubMed Google Scholar Males are more frequently affected (2:1 male to female ratio), and such a finding is consistent across ethnic groups.4Chung C.S. Nemechek R.W. Larsen I.J. Ching G.H. Genetic and epidemiological studies of clubfoot in Hawaii. General and medical considerations.Hum. Hered. 1969; 19: 321-342Crossref PubMed Scopus (66) Google Scholar Clubfoot is bilateral in approximately 50% of all cases, and the right foot is more often affected in unilateral cases.3Lochmiller C. Johnston D. Scott A. Risman M. Hecht J.T. Genetic epidemiology study of idiopathic talipes equinovarus.Am. J. Med. Genet. 1998; 79: 90-96Crossref PubMed Scopus (135) Google Scholar Limb patterning and growth are carefully regulated through a complex network of transcription-factor and signaling-molecule expression.6Zelzer E. Olsen B.R. The genetic basis for skeletal diseases.Nature. 2003; 423: 343-348Crossref PubMed Scopus (210) Google Scholar, 7Tickle C. Patterning systems–from one end of the limb to the other.Dev. Cell. 2003; 4: 449-458Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar Two transcription-factor genes, Pitx1 (MIM 602149) and Tbx4 (MIM 601719), are expressed predominantly in the developing hindlimb and are only minimally expressed in the forelimb,8Szeto D.P. Ryan A.K. O'Connell S.M. Rosenfeld M.G. P-OTX: A PIT-1-interacting homeodomain factor expressed during anterior pituitary gland development.Proc. Natl. Acad. Sci. USA. 1996; 93: 7706-7710Crossref PubMed Scopus (238) Google Scholar, 9Lanctot C. Lamolet B. Drouin J. The bicoid-related homeoprotein Ptx1 defines the most anterior domain of the embryo and differentiates posterior from anterior lateral mesoderm.Development. 1997; 124: 2807-2817PubMed Google Scholar, 10Gibson-Brown J.J. Agulnik S.I. Chapman D.L. Alexiou M. Garvey N. Silver L.M. Papaioannou V.E. Evidence of a role for T-box genes in the evolution of limb morphogenesis and the specification of forelimb/hindlimb identity.Mech. Dev. 1996; 56: 93-101Crossref PubMed Scopus (230) Google Scholar suggesting that they may be important regulators of hindlimb identity. In support of this, studies have shown that misexpression of Pitx1 in the developing chick wing bud changes the morphology and digit number such that the limb more resembles a leg.11Logan M. Tabin C.J. Role of Pitx1 upstream of Tbx4 in specification of hindlimb identity.Science. 1999; 283: 1736-1739Crossref PubMed Scopus (227) Google Scholar, 12Szeto D.P. Rodriguez-Esteban C. Ryan A.K. O'Connell S.M. Liu F. Kioussi C. Gleiberman A.S. Izpisua-Belmonte J.C. Rosenfeld M.G. Role of the Bicoid-related homeodomain factor Pitx1 in specifying hindlimb morphogenesis and pituitary development.Genes Dev. 1999; 13: 484-494Crossref PubMed Scopus (329) Google Scholar Likewise, loss of Pitx1 expression in the developing mouse causes the hindlimb to assume morphology and growth features of the corresponding bones in the forelimb.12Szeto D.P. Rodriguez-Esteban C. Ryan A.K. O'Connell S.M. Liu F. Kioussi C. Gleiberman A.S. Izpisua-Belmonte J.C. Rosenfeld M.G. Role of the Bicoid-related homeodomain factor Pitx1 in specifying hindlimb morphogenesis and pituitary development.Genes Dev. 1999; 13: 484-494Crossref PubMed Scopus (329) Google Scholar, 13Lanctot C. Moreau A. Chamberland M. Tremblay M.L. Drouin J. Hindlimb patterning and mandible development require the Ptx1 gene.Development. 1999; 126: 1805-1810Crossref PubMed Google Scholar Furthermore, alterations in PITX1 expression underlie rapid evolutionary changes in pelvic morphology in vertebrate populations, including stickleback fish and manatee,14Shapiro M.D. Marks M.E. Peichel C.L. Blackman B.K. Nereng K.S. Jonsson B. Schluter D. Kingsley D.M. Genetic and developmental basis of evolutionary pelvic reduction in threespine sticklebacks.Nature. 2004; 428: 717-723Crossref PubMed Scopus (650) Google Scholar, 15Shapiro M.D. Bell M.A. Kingsley D.M. Parallel genetic origins of pelvic reduction in vertebrates.Proc. Natl. Acad. Sci. USA. 2006; 103: 13753-13758Crossref PubMed Scopus (174) Google Scholar supporting an important role for PITX1 in hindlimb development. The opportunity to identify a gene responsible for idiopathic clubfoot arose from the identification of a five-generation North American family of European descent in which clubfoot segregates as an autosomal-dominant condition with reduced penetrance. In addition to bilateral clubfoot, the proband also manifests bilateral foot preaxial polydactyly, right-sided tibial hemimelia (Figures 1A and 1B), and a left-sided small preauricular skin tag (not shown). Five additional family members are affected with clubfoot; three of whom have increased severity on the right. One male has unilateral left clubfoot. Two individuals have bilateral oblique talus, manifesting as pes planus (flatfoot). These two patients had never been treated or evaluated, whereas all six individuals with clubfoot underwent either surgical treatment or serial casting by an orthopaedic surgeon who diagnosed their condition as clubfoot. Three individuals have bilaterally hypoplastic patella resulting in patellar dislocations in late childhood. One individual had developmental hip dysplasia, but he was born in breech presentation, a known risk factor for this condition. He also has pes planus. Other than the proband, no other family members have polydactyly or tibial hemimelia. No upper extremity abnormalities or dysmorphic craniofacial features were noted. There are five clinically unaffected female carriers and no clinically unaffected males. After informed consent and approval were obtained from the local human ethics review committees, a genome-wide linkage scan was performed on 13 family members with Affymetrix 10K mapping SNP genotypes. Linkage was performed assuming autosomal-dominant inheritance with 70% penetrance, a disease allele frequency of 0.01%, no phenocopies, and Affymetrix "Caucasian" allele frequencies. Parametric multipoint logarithm of the odds (LOD) score analysis performed with sets of 60–100 markers with GENEHUNTER16Kruglyak L. Daly M.J. Reeve-Daly M.P. Lander E.S. Parametric and nonparametric linkage analysis: A unified multipoint approach.Am. J. Hum. Genet. 1996; 58: 1347-1363PubMed Google Scholar yielded a maximum LOD score of 3.31 with markers on chromosome 5q23.3-q31.2 (Figure 2). Recombinants delineated the critical genetic region to a 9 cM interval between SNP_A-1507970 and SNP_A-1517278, corresponding to a 12 Mb region (chr5:126,890,333-138,147,299) (Figure 3). The chromosome 5q23.3-q31.2 candidate region contains more than 80 genes.Figure 3Haplotype Analysis Indicating Markers on Chromosome 5q23.3-q31.2 Common to All Affected IndividualsShow full captionHaplotypes were created with GENEHUNTER and viewed on Haplopainter.43Thiele H. Nurnberg P. HaploPainter: A tool for drawing pedigrees with complex haplotypes.Bioinformatics. 2005; 21: 1730-1732Crossref PubMed Scopus (217) Google Scholar Presence or absence of the PITX1 E130K mutation is noted with (+) or (−). The arrow indicates the proband.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Haplotypes were created with GENEHUNTER and viewed on Haplopainter.43Thiele H. Nurnberg P. HaploPainter: A tool for drawing pedigrees with complex haplotypes.Bioinformatics. 2005; 21: 1730-1732Crossref PubMed Scopus (217) Google Scholar Presence or absence of the PITX1 E130K mutation is noted with (+) or (−). The arrow indicates the proband. Sequencing of PITX1, a bicoid-related homeodomain gene located within the candidate interval, revealed a single missense nucleotide change c.388G→A that results in the substitution of lysine for glutamic acid (E130K) in the highly conserved homeodomain (Figure 4A). This glutamic acid is at position 42 of the homeodomain within the DNA-binding third helix and is located near the Lys50 residue that imparts bicoid-type DNA-binding specificity.17Hanes S.D. Brent R. DNA specificity of the bicoid activator protein is determined by homeodomain recognition helix residue 9.Cell. 1989; 57: 1275-1283Abstract Full Text PDF PubMed Scopus (377) Google Scholar The PITX1 E130K mutation was present in all affected family members as well as in two tested obligate carriers but was absent in 500 white North American control subjects. On the basis of the NMR solution structure of the PITX homeodomain bound to DNA, the glutamic-acid side chain at position 42 (corresponding to the E130K mutation) extends into the major groove of DNA when bound18Chaney B.A. Clark-Baldwin K. Dave V. Ma J. Rance M. Solution structure of the K50 class homeodomain PITX2 bound to DNA and implications for mutations that cause Rieger syndrome.Biochemistry. 2005; 44: 7497-7511Crossref PubMed Scopus (42) Google Scholar (Figure 4B). Glutamic acid at position 42 is conserved in all human PITX family members, across species, and in other nonbicoid homeodomain genes, including PAX7 (Figure 4C). To characterize the effects of the PITX1 E130K substitution on transcription-factor activity, we measured its ability to transactivate a luciferase reporter consisting of a thymidine kinase promoter preceded by four bicoid-binding sites (TAATCC).19Amendt B.A. Sutherland L.B. Semina E.V. Russo A.F. The molecular basis of Rieger syndrome. Analysis of Pitx2 homeodomain protein activities.J. Biol. Chem. 1998; 273: 20066-20072Crossref PubMed Scopus (116) Google Scholar COS7 cells were used for these experiments because they do not express PITX1 endogenously. The PITX1 E130K mutation did not affect its expression or nuclear localization in transiently transfected COS7 cells (Figures 5A and 5B). Wild-type (WT) PITX1 transactivated a luciferase reporter gene ∼7-fold, whereas the PITX1 E130K mutant activated the same reporter only ∼4-fold (Figure 5C), demonstrating reduced transactivation activity. Because bicoid-related homeodomain proteins form functional homodimers,20Saadi I. Kuburas A. Engle J.J. Russo A.F. Dominant negative dimerization of a mutant homeodomain protein in Axenfeld-Rieger syndrome.Mol. Cell. Biol. 2003; 23: 1968-1982Crossref PubMed Scopus (31) Google Scholar we examined whether the PITX1 E130K mutant might exert dominant-negative inhibition of WT PITX1 transactivation. Cotransfection of equal amounts of PITX1 E130K plasmid DNA with WT PITX1 reduced the ability of WT PITX1 to activate the luciferase reporter in COS7 cells (Figure 5C, lane 4). Negative effects of PITX1 E130K mutant expression were also demonstrated on endogenous PITX1 in HeLa cells (data not shown). A dose-response curve demonstrated that the addition of increasing quantities of transfected WT PITX1 DNA had no effect on luciferase activation, but increasing PITX1 E130K DNA resulted in a dose-dependent decrease in luciferase activity (Figure 5D), supporting a dominant-negative effect of the PITX1 E130K mutant protein on transcriptional activation by PITX1. Alterations of PITX1 expression have been shown to underlie evolutionary adaptation of vertebrate hindlimb structures,14Shapiro M.D. Marks M.E. Peichel C.L. Blackman B.K. Nereng K.S. Jonsson B. Schluter D. Kingsley D.M. Genetic and developmental basis of evolutionary pelvic reduction in threespine sticklebacks.Nature. 2004; 428: 717-723Crossref PubMed Scopus (650) Google Scholar, 15Shapiro M.D. Bell M.A. Kingsley D.M. Parallel genetic origins of pelvic reduction in vertebrates.Proc. Natl. Acad. Sci. USA. 2006; 103: 13753-13758Crossref PubMed Scopus (174) Google Scholar but this report is to our knowledge the first evidence for PITX1 mutation in human disease. Exclusive involvement of the lower limb in individuals with the PITX1 E130K mutation is consistent with the preferential expression of PITX1 in the hindlimb8Szeto D.P. Ryan A.K. O'Connell S.M. Rosenfeld M.G. P-OTX: A PIT-1-interacting homeodomain factor expressed during anterior pituitary gland development.Proc. Natl. Acad. Sci. USA. 1996; 93: 7706-7710Crossref PubMed Scopus (238) Google Scholar, 9Lanctot C. Lamolet B. Drouin J. The bicoid-related homeoprotein Ptx1 defines the most anterior domain of the embryo and differentiates posterior from anterior lateral mesoderm.Development. 1997; 124: 2807-2817PubMed Google Scholar and its known role in hindlimb development.11Logan M. Tabin C.J. Role of Pitx1 upstream of Tbx4 in specification of hindlimb identity.Science. 1999; 283: 1736-1739Crossref PubMed Scopus (227) Google Scholar, 12Szeto D.P. Rodriguez-Esteban C. Ryan A.K. O'Connell S.M. Liu F. Kioussi C. Gleiberman A.S. Izpisua-Belmonte J.C. Rosenfeld M.G. Role of the Bicoid-related homeodomain factor Pitx1 in specifying hindlimb morphogenesis and pituitary development.Genes Dev. 1999; 13: 484-494Crossref PubMed Scopus (329) Google Scholar, 13Lanctot C. Moreau A. Chamberland M. Tremblay M.L. Drouin J. Hindlimb patterning and mandible development require the Ptx1 gene.Development. 1999; 126: 1805-1810Crossref PubMed Google Scholar Other known genetic causes of tibial hemimelia were also excluded in this family, including mutations in the long-distance sonic hedgehog enhancer21Lettice L.A. Heaney S.J. Purdie L.A. Li L. de Beer P. Oostra B.A. Goode D. Elgar G. Hill R.E. de Graaff E. A long-range Shh enhancer regulates expression in the developing limb and fin and is associated with preaxial polydactyly.Hum. Mol. Genet. 2003; 12: 1725-1735Crossref PubMed Scopus (748) Google Scholar, 22Zguricas J. Heus H. Morales-Peralta E. Breedveld G. Kuyt B. Mumcu E.F. Bakker W. Akarsu N. Kay S.P. Hovius S.E. et al.Clinical and genetic studies on 12 preaxial polydactyly families and refinement of the localisation of the gene responsible to a 1.9 cM region on chromosome 7q36.J. Med. Genet. 1999; 36: 32-40PubMed Google Scholar and duplications of the SHFM3 locus on chromosome 10q24-q25,23Nunes M.E. Schutt G. Kapur R.P. Luthardt F. Kukolich M. Byers P. Evans J.P. A second autosomal split hand/split foot locus maps to chromosome 10q24-q25.Hum. Mol. Genet. 1995; 4: 2165-2170Crossref PubMed Scopus (73) Google Scholar although both of these disorders are also associated with upper extremity involvement. Additional evidence that the PITX1 E130K mutation is responsible for these lower-extremity birth defects is shown by the high degree of evolutionary conservation at the site of the mutation within the critically important DNA-binding homeodomain, as well as by the negative functional effects of this mutation on transcriptional activity. Few transcriptional targets of PITX1 are known in the developing limb, with the exception of TBX4,11Logan M. Tabin C.J. Role of Pitx1 upstream of Tbx4 in specification of hindlimb identity.Science. 1999; 283: 1736-1739Crossref PubMed Scopus (227) Google Scholar a transcription factor that is likewise preferentially expressed in the lower limb.10Gibson-Brown J.J. Agulnik S.I. Chapman D.L. Alexiou M. Garvey N. Silver L.M. Papaioannou V.E. Evidence of a role for T-box genes in the evolution of limb morphogenesis and the specification of forelimb/hindlimb identity.Mech. Dev. 1996; 56: 93-101Crossref PubMed Scopus (230) Google Scholar Several phenotypic similarities are present between patients with small patella syndrome (MIM 147891) caused by TBX4 mutations24Bongers E.M. Duijf P.H. van Beersum S.E. Schoots J. Van Kampen A. Burckhardt A. Hamel B.C. Losan F. Hoefsloot L.H. Yntema H.G. et al.Mutations in the human TBX4 gene cause small patella syndrome.Am. J. Hum. Genet. 2004; 74: 1239-1248Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar and patients described here with PITX1 mutations, including patellar hypoplasia and pes planus. Asymmetric involvement has not been described in small patella syndrome. Characteristic foot abnormalities, including shortened fourth and fifth rays and wide spacing between the first and second toes, commonly seen with TBX4 mutations,25Bongers E.M. Van Bokhoven H. Van Thienen M.N. Kooyman M.A. Van Beersum S.E. Boetes C. Knoers N.V. Hamel B.C. The small patella syndrome: Description of five cases from three families and examination of possible allelism with familial patella aplasia-hypoplasia and nail-patella syndrome.J. Med. Genet. 2001; 38: 209-214Crossref PubMed Google Scholar appear to be uncommon in individuals with the PITX1 E130K mutation. Variable expressivity is common in both genetic disorders. Mutations in all three known PITX bicoid-related homeobox-gene family members result in autosomal-dominant human congenital disorders. PITX2 mutations were the first to be described in individuals with Rieger syndrome,26Semina E.V. Reiter R.S. Murray J.C. Isolation of a new homeobox gene belonging to the Pitx/Rieg family: Expression during lens development and mapping to the aphakia region on mouse chromosome 19.Hum. Mol. Genet. 1997; 6: 2109-2116Crossref PubMed Scopus (160) Google Scholar a disorder characterized by ocular anterior chamber anomalies, craniofacial dysmorphisms, dental hypoplasia, and umbilical stump abnormalities, whereas PITX3 mutations result in autosomal-dominant cataracts and anterior segment mesenchymal dysgenesis.27Semina E.V. Ferrell R.E. Mintz-Hittner H.A. Bitoun P. Alward W.L. Reiter R.S. Funkhauser C. Daack-Hirsch S. Murray J.C. A novel homeobox gene PITX3 is mutated in families with autosomal-dominant cataracts and ASMD.Nat. Genet. 1998; 19: 167-170Crossref PubMed Scopus (317) Google Scholar Although many missense and nonsense PITX2 mutations are presumed to cause Rieger syndrome through a loss of function,26Semina E.V. Reiter R.S. Murray J.C. Isolation of a new homeobox gene belonging to the Pitx/Rieg family: Expression during lens development and mapping to the aphakia region on mouse chromosome 19.Hum. Mol. Genet. 1997; 6: 2109-2116Crossref PubMed Scopus (160) Google Scholar a dominant-negative mutation was previously described (K88E) at homeodomain position 50,28Saadi I. Semina E.V. Amendt B.A. Harris D.J. Murphy K.P. Murray J.C. Russo A.F. Identification of a dominant negative homeodomain mutation in Rieger syndrome.J. Biol. Chem. 2001; 276: 23034-23041Crossref PubMed Scopus (69) Google Scholar which is located near the PITX1 E130K mutation (homeodomain position 42) described here. Asymmetric involvement is a hallmark of altered PITX1 expression, as shown by the right-sided predominance of human lower-extremity malformations caused by the PITX1 E130K mutation. Similarly, loss of PITX1 expression in stickleback fish results in a greater reduction of pelvic structures on the right.14Shapiro M.D. Marks M.E. Peichel C.L. Blackman B.K. Nereng K.S. Jonsson B. Schluter D. Kingsley D.M. Genetic and developmental basis of evolutionary pelvic reduction in threespine sticklebacks.Nature. 2004; 428: 717-723Crossref PubMed Scopus (650) Google Scholar Manatee vestigial pelvic structures also show left-right directional asymmetry, suggesting that similar evolutionary mechanisms were responsible for the transition of this marine mammal from a four-legged terrestrial ancestor.15Shapiro M.D. Bell M.A. Kingsley D.M. Parallel genetic origins of pelvic reduction in vertebrates.Proc. Natl. Acad. Sci. USA. 2006; 103: 13753-13758Crossref PubMed Scopus (174) Google Scholar Loss of Pitx1 expression in mice also causes greater shortening of femur length and loss of digits on the right foot as compared to the left.12Szeto D.P. Rodriguez-Esteban C. Ryan A.K. O'Connell S.M. Liu F. Kioussi C. Gleiberman A.S. Izpisua-Belmonte J.C. Rosenfeld M.G. Role of the Bicoid-related homeodomain factor Pitx1 in specifying hindlimb morphogenesis and pituitary development.Genes Dev. 1999; 13: 484-494Crossref PubMed Scopus (329) Google Scholar, 13Lanctot C. Moreau A. Chamberland M. Tremblay M.L. Drouin J. Hindlimb patterning and mandible development require the Ptx1 gene.Development. 1999; 126: 1805-1810Crossref PubMed Google Scholar This directional asymmetry has been ascribed to the unmasking of residual PITX2 activity resulting from the loss of PITX1 expression.29Marcil A. Dumontier E. Chamberland M. Camper S.A. Drouin J. Pitx1 and Pitx2 are required for development of hindlimb buds.Development. 2003; 130: 45-55Crossref PubMed Scopus (135) Google Scholar PITX2 belongs to a small group of genes important for right-left body-axis patterning that results from its preferential expression in left lateral-plate mesoderm.30Logan M. Pagan-Westphal S.M. Smith D.M. Paganessi L. Tabin C.J. The transcription factor Pitx2 mediates situs-specific morphogenesis in response to left-right asymmetric signals.Cell. 1998; 94: 307-317Abstract Full Text Full Text PDF PubMed Scopus (364) Google Scholar, 31Ryan A.K. Blumberg B. Rodriguez-Esteban C. Yonei-Tamura S. Tamura K. Tsukui T. de la Pena J. Sabbagh W. Greenwald J. Choe S. et al.Pitx2 determines left-right asymmetry of internal organs in vertebrates.Nature. 1998; 394: 545-551Crossref PubMed Scopus (418) Google Scholar, 32Piedra M.E. Icardo J.M. Albajar M. Rodriguez-Rey J.C. Ros M.A. Pitx2 participates in the late phase of the pathway controlling left-right asymmetry.Cell. 1998; 94: 319-324Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar, 33Yoshioka H. Meno C. Koshiba K. Sugihara M. Itoh H. Ishimaru Y. Inoue T. Ohuchi H. Semina E.V. Murray J.C. et al.Pitx2, a bicoid-type homeobox gene, is involved in a lefty-signaling pathway in determination of left-right asymmetry.Cell. 1998; 94: 299-305Abstract Full Text Full Text PDF PubMed Scopus (309) Google Scholar Asymmetric lower-limb reduction malformations have frequently been described in familial tibial hemimelia, in which the right side is nearly always preferentially affected.34Wiedemann H.R. Opitz J.M. Brief clinical report: Unilateral partial tibia defect with preaxial polydactyly, general micromelia, and trigonomacrocephaly with a note on "developmental resistance".Am. J. Med. Genet. 1983; 14: 467-471Crossref PubMed Scopus (12) Google Scholar, 35Clark M.W. Autosomal dominant inheritance of tibial meromelia. Report of a kindred.J. Bone Joint Surg. Am. 1975; 57: 262-264PubMed Google Scholar The consistent asymmetry noted in tibial hemimelia prompted Wiedemann and Opitz to propose a mechanism of "developmental resistance"34Wiedemann H.R. Opitz J.M. Brief clinical report: Unilateral partial tibia defect with preaxial polydactyly, general micromelia, and trigonomacrocephaly with a note on "developmental resistance".Am. J. Med. Genet. 1983; 14: 467-471Crossref PubMed Scopus (12) Google Scholar in which the left lower limb was protected from the effect of the mutation. Although vascular differences between left and right umbilical vessels have also been proposed as a possible mechanism leading to directional asymmetry,36Searle A. The genetics and morphology of two "luxoid" mutants in the house mouse.Genet. Res. Camb. 1964; 5: 171-197Crossref Scopus (60) Google Scholar molecular differences between limbs may account for most of this asymmetry.37Schreiner C.M. Scott Jr., W.J. Supp D.M. Potter S.S. Correlation of forelimb malformation asymmetries with visceral organ situs in the transgenic mouse insertional mutation, legless.Dev. Biol. 1993; 158: 560-562Crossref PubMed Scopus (22) Google Scholar Relative levels of PITX1 and PITX2 expression in the developing limb may be the first example of such a molecular signal that contributes to the overall propensity for right-sided malformations in an affected family or population. However, on an individual basis, other environmental or genetic effects may overcome this tendency, as demonstrated by the unilateral left-sided clubfoot seen in one individual with the PITX1 E130K mutation. Occasional stickleback fish with left-sided pelvic-spine reduction and PITX1 loss have also been described.15Shapiro M.D. Bell M.A. Kingsley D.M. Parallel genetic origins of pelvic reduction in vertebrates.Proc. Natl. Acad. Sci. USA. 2006; 103: 13753-13758Crossref PubMed Scopus (174) Google Scholar, 38Coyle S.M. Huntingford F.A. Peichel C.L. Parallel evolution of Pitx1 underlies pelvic reduction in Scottish threespine stickleback (Gasterosteus aculeatus).J. Hered. 2007; 98: 581-586Crossref PubMed Scopus (47) Google Scholar Several clinical characteristics of individuals with the PITX1 E130K mutation suggest that PITX1 and/or its pathways may be involved in the etiology of idiopathic clubfoot. First, the majority of the affected individuals in this family have isolated clubfoot with no other abnormalities. Second, incomplete penetrance was noted with the presence of five carrier females; such a finding is consistent with the lower incidence of idiopathic clubfoot in females.3Lochmiller C. Johnston D. Scott A. Risman M. Hecht J.T. Genetic epidemiology study of idiopathic talipes equinovarus.Am. J. Med. Genet. 1998; 79: 90-96Crossref PubMed Scopus (135) Google Scholar Third, tibial hemimelia and clubfoot affect the right foot more frequently,3Lochmiller C. Johnston D. Scott A. Risman M. Hecht J.T. Genetic epidemiology study of idiopathic talipes equinovarus.Am. J. Med. Genet. 1998; 79: 90-96Crossref PubMed Scopus (135) Google Scholar, 35Clark M.W. Autosomal dominant inheritance of tibial meromelia. Report of a kindred.J. Bone Joint Surg. Am. 1975; 57: 262-264PubMed Google Scholar, 39Kalamchi A. Dawe R.V. Congenital deficiency of the tibia.J. Bone Joint Surg. Br. 1985; 67: 581-584PubMed Google Scholar suggesting the possibility that PITX1 or its pathways may contribute to this directional asymmetry. However, we were unable to identify any additional PITX1 coding mutations in 100 patients with clubfoot and eight with tibial hemimelia, including probands from three separate multigenerational families in which exclusively right-sided lower-limb malformations segregate. Failure to identify additional mutations in our study was not entirely unexpected, as studies in stickleback fish also failed to demonstrate PITX1 mutations, despite strong linkage to the region and evidence of altered gene expression.14Shapiro M.D. Marks M.E. Peichel C.L. Blackman B.K. Nereng K.S. Jonsson B. Schluter D. Kingsley D.M. Genetic and developmental basis of evolutionary pelvic reduction in threespine sticklebacks.Nature. 2004; 428: 717-723Crossref PubMed Scopus (650) Google Scholar Many highly conserved noncoding regions potentially corresponding to limb-specific regulatory elements occur within the >300 kb gene desert surrounding PITX1 and mutations in these regions may result in a similar phenotype. It is also possible that mutations in genes regulating PITX1 expression or other early expressed genes involved in establishing left-right asymmetry40Hamada H. Meno C. Watanabe D. Saijoh Y. Establishment of vertebrate left-right asymmetry.Nat. Rev. Genet. 2002; 3: 103-113Crossref PubMed Scopus (428) Google Scholar may cause asymmetric lower-limb malformations. Our results demonstrate a role for the bicoid-related homeobox gene PITX1 in a variety of human lower-limb malformations, including clubfoot, pes planus, tibial hemimelia, and patellar hypoplasia. The asymmetric hallmark of altered PITX1 expression seen in vertebrates as diverse as humans, mice, stickleback fish, and manatee supports the possibility that PITX1 or its pathways may be etiologically responsible for the increased incidence of right-sided tibial hemimelia and clubfoot. Furthermore, implication of a transcription-factor gene involved in early limb development suggests additional pathways for the future investigation of idiopathic clubfoot etiology in humans. We kindly thank Dr. Andrew Russo for use of the TK-Bic luciferase reporter plasmid and the National Institutes of Health Neuroscience Microarray Consortium for performing the Affymetrix microarray analyses. The PITX1 sequence annotation used here corresponds to NM_002653.4 coding sequence only. This work was supported by grants from the National Institutes of Health (K12 HD001459), The Children's Discovery Institute, and March of Dimes Basil O'Connor Starter Scholar Research Award, St Louis Children's Hospital Foundation, Pediatric Orthopaedic Society of North America, and Shriners Hospital. The authors have no conflict of interest to disclose. The URLs for data presented herein are as follows:Mulitple sequence Alignment by CLUSTALW, http://align.genome.jpOnline Mendelian Inheritance of Man (OMIM), http://www.ncbi.nlm.nih.gov/OmimSwiss PDB DeepView program, http://ca.expasy.org/spdbvUCSC Genome Browser, http://genome.ucsc.edu/cgi-bin/hgGateway
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