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

We recently reported the positional cloning of a homeobox gene involved in the pathogenesis of Rieger syndrome, RIEG1 , and its mouse homolog, Rieg1 . Rieg1 (also independently described as Pitx2) is highly homologous to the Ptx1/Potx gene product, suggesting that there may be additional members of this novel Pitx family. The Pitx genes play an important role in eye, tooth, pituitary and umbilical region development as evidenced by Rieger syndrome and iris hypoplasia phenotypes, resulting from mutations in the RIEG1 gene and by expression studies. In order to characterize further the Pitx gene family we searched mouse cDNA libraries to identify additional members. A new gene was isolated which encodes a homeoprotein with strong homology to the other Pitx proteins and 97-100% identity in the homeodomain itself, suggesting that this is a third member of the family, Pitx3 . In whole mount in situ hybridization on mouse embryos ranging from 8.5 to 11.5 days post-coitum (d.p.c.), Pitx3 mRNA was seen only in the developing lens starting at day 11. Hybridization on cross- sections revealed strong signals in the lens vesicle in 11 d.p.c. embryos and throughout the lens, particularly in the anterior epithelium and equator region in 15 d.p.c. embryos. Pitx3 was mapped close to aphakia on mouse chromosome 19. The aphakia homozygous mouse is characterized by small eyes lacking a lens, which fail to develop beyond 11 d.p.c. These data make Pitx3 a strong candidate gene for the aphakia phenotype in the mouse and suggest a role for the human homolog in congenital lens malformations.      

Expression of the homeobox gene Pitx2 in neural crest is required for optic stalk and ocular anterior segment development

Heterozygous mutations in the homeobox gene, PITX2, result in ocular anterior segment defects and a high incidence of early-onset glaucoma. Pitx2 is expressed in both the neural crest and the mesoderm-derived precursors of the periocular mesenchyme. Complete loss of function in mice results in agenesis or severe disruption of periocular mesenchyme structures and extrinsic defects in early optic nerve development. However, the specific requirements for Pitx2 in neural crest versus mesoderm could not be determined using these mice, and only roles in the initial stages of eye development could be assessed due to early embryonic lethality. To determine the specific roles of Pitx2 in the neural crest precursor pool, we generated neural crest-specific Pitx2 knockout mice (Pitx2-ncko). Because Pitx2-nkco mice are viable, we also analyzed gene function in later eye development. Pitx2 is intrinsically required in neural crest for specification of corneal endothelium, corneal stroma and the sclera. Pitx2 function in neural crest is also required for normal development of ocular blood vessels. Pitx2-ncko mice exhibit a unique optic nerve phenotype in which the eyes are progressively displaced towards the midline until they are directly attached to the ventral hypothalamus. As Pitx2 is not expressed in the optic stalk, an essential function of PITX2 protein in neural crest is to regulate an extrinsic factor(s) required for development of the optic nerve. We propose a revised model of optic nerve development and new mechanisms that may underlie the etiology of glaucoma in Axenfeld-Rieger patients.     

垂体同源盒家族因子3基因在大鼠脊髓发育和生存维持过程中的表达变化

目的:探讨垂体同源盒家族因子3(Pitx3)基因在SD大鼠脊髓不同年龄阶段的表达变化以及细胞定位。方法:采用半定量RT-PCR和Western Blot检测不同年龄组大鼠脊髓中Pitx3 mRNA及蛋白水平表达变化;采用免疫荧光和免疫组化染色显示Pitx3在脊髓中的细胞定位。结果:(1)RT-PCR和Western Blot检测显示Pitx3基因在各年龄组脊髓各段均有表达;大鼠脊髓发育过程中Pitx3 mRNA及其蛋白水平出生前逐渐升高,在出生后(P0 d)达高锋,以后维持在一定的生理水平,且出生以后随年龄的增长表达呈下调趋势;(2)免疫荧光双标证实Pitx3蛋白在P0 d大鼠脊髓主要定位于脊髓灰质,与神经元的标记物NeuN存在共定位;免疫组化检测显示Pitx3蛋白集中分布于各年龄组脊髓灰质腹角运动神经元胞质中,少量分布于背角和侧角神经元胞质中。结论:在大鼠脊髓发育和生存维持过程中,Pitx3在基因和蛋白水平呈现明显的时相变化;提示Pitx3基因的表达在脊髓腹角运动神经元的发育和生存维持中可能发挥一定的调控作用。     

Candidate gene sequencing of LHX2, HESX1, and SOX2 in a large schizencephaly cohort

Schizencephaly is a malformation of cortical development characterized by gray matter-lined clefts in the cerebral cortex and a range of neurological presentations. In some cases, there are features of septo-optic dysplasia concurrently with schizencephaly. The etiologies of both schizencephaly and septo-optic dysplasia are thought to be heterogeneous, but there is evidence that at least some cases have genetic origin. We hypothesized that these disorders may be caused by mutations in three candidate genes: LHX2, a gene with an important cortical patterning role, and HESX1 and SOX2, genes that have been associated with septo-optic dysplasia. We sequenced a large cohort of patients with schizencephaly, some with features of septo-optic dysplasia, for mutations in these genes. No pathogenic mutations were observed, suggesting that other genes or non-genetic factors influencing genes critical to brain development must be responsible for schizencephaly.     

The Pitx2 protein in mouse development.

The Rieger syndrome, an autosomal dominant disorder involving ocular, dental, and umbilical defects is caused by mutations in PITX2, a Bicoid-type homeobox protein. Mouse Pitx2 mRNA is expressed in eye, tooth and umbilicus consistent with the human Riegers phenotype. Moreover, Pitx2 is involved in the Nodal/Sonic hedgehog pathway that determines left/right polarity. In this report we demonstrate a 32-kDa polypeptide on Western blots of nuclear extracts from a rat pituitary cell line, using a Pitx2 specific antibody (designated P2R10). We describe also for the first time expression of the Pitx2 protein in mouse. Pitx2 protein immunostaining was detectable during the development of the eye, tooth, umbilicus, and also in the pituitary, heart, gut, and limb. We demonstrate for the first time directly that Pitx2 is asymmetrically expressed in early heart, gut, and lung development.     

Novel germline BRCA1 and BRCA2 mutations in breast and breast/ovarian cancer families from the Czech Republic

Germline mutations in breast cancer susceptibility genes, BRCA1 and BRCA2, are responsible for a substantial proportion of high‐risk breast and breast/ovarian cancer families. To characterize the spectrum of BRCA1 and BRCA2 mutations, we screened Czech families with breast/ovarian cancer using the non‐radioactive protein truncation test, heteroduplex analysis and direct sequencing. In a group of 100 high‐risk breast and breast/ovarian cancer families, four novel frame shift mutations were identified in BRCA1 and BRCA2 genes. In BRCA1, two novel frame shift mutations were identified as 3761‐3762delGA and 2616‐2617ins10; in BRCA2, two novel frame shift mutations were identified as 5073‐5074delCT and 6866delC. Furthermore, a novel missense substitution M18K in BRCA1 gene in a breast/ovarian cancer family was identified which lies adjacent just upstream of the most highly conserved C3HC4 RING zinc finger motif. To examine the tertiary structure of the RING zinc finger domain and possible effects of M18K substitution on its stability, we used threading techniques according to the crystal structure of RAG1 dimerization domain of the DNA‐binding protein. © 2000 Wiley‐Liss, Inc.     

Phenotype variation and newcomers in ion channel disorders

Ion channels are part of a large family of macromolecules whose functions include the control and maintenance of electrical potential across cell membranes, secretion and signal transduction. Close inspection of the physiological processes involved in channel function and the secondary structure of various ion channels has served as a basis for subdividing ion channels into a number of superfamilies. The voltage-gated ion channels are one of these superfamilies. Recent work has shown that mutations in various ion channel genes are responsible for a number of neuromuscular and neurological disorders. Correlation of the various mutations with the clinical phenotype is providing us with insight into the pathophysiology of these channel proteins. Interestingly, different mutations within the same gene may cause quite distinct clinical disorders, while mutations in different channel genes may result in very similar phenotypes (genetic heterogeneity). Examples of phenotypic variation and genetic heterogeneity are presented in the context of the periodic paralytic disorders of skeletal muscle, episodic ataxia, migraine, long QT syndrome and paroxysmal dyskinesia. Some of these disorders are known to be caused by mutations in ion channel genes, while in the episodic movement disorders, ion channel genes are considered excellent candidate genes.      

Reciprocal effect of Waardenburg syndrome mutations on DNA binding by the Pax-3 paired domain and homeodomain

The Pax-3 protein contains two DNA-binding domains, a paired domain and a homeodomain. Mutations in Pax-3 cause Waardenburg syndrome (WS) in humans and the mouse Splotch (Sp) phenotype. In the Sp-delayed mouse, a mutation in the Pax-3 paired domain (G9R) abrogates the DNA-binding activity of both the paired domain and the homeodomain, suggesting that they may functionally interact. To investigate this possibility further, we have analyzed the DNA-binding properties of additional point mutants in the Pax-3 paired domain and homeodomain that occur in WS patients (F12L, N14H, G15S, P17L, R23L, G48A, S51F and G66D in the paired domain, V47F and R53G in the homeodomain), the Pax-1 un mutation (G15A) and a substitution associated with Peters' anomaly in the PAX-6 gene (R23G). Within the paired domain, seven of 10 mutations were found to abrogate DNA-binding by the paired domain. Remarkably, these seven mutations also affected DNA binding by the homeodomain, causing either a complete loss (P17L and G66D), a reduction (R23G, R23L, G15S and G15A) or an increase in DNA-binding activity (N14H). In addition, the effect of paired domain mutations occurred at the level of monomer formation by the homeodomain, while the dimerization potential of this domain seemed unaffected in mutants where it could be analyzed. Furthermore, while both homeodomain mutations were found to abolish DNA binding by this domain, the R53G mutation also abrogated DNA binding by the paired domain. The important observation that independent mutations in either domain can affect DNA binding by the other in the intact Pax- 3 protein strongly suggests that the two domains are not functionally independent but bind DNA through cooperative interactions. Modeling the deleterlous mutations on the three-dimensional structure of the paired domain of Drosophila Prd shows that these mutations cluster at the DNA interface, thus suggesting that a series of DNA contacts are essential for DNA binding by both the paired domain and the homeodomain of Pax-3.      

Pitx2c overexpression promotes cell proliferation and arrests differentiation in myoblasts.

Pitx2 is a paired-related homeobox gene that has been shown to play a central role during development. In the mouse, there are three isoforms, Pitx2a, b, and c, which differ only in their amino terminal regions. Pitx2 is expressed in myotomes, myoblasts, and myofibers and may be involved in muscle patterning. However, the mechanism by which Pitx2 acts in muscle cell lineages as well as the distinct functions of the individual isoforms have not been investigated. In this study, we used Sol8 myoblasts to investigate the function of Pitx2 in skeletal myogenesis. We found that Pitx2c is the main Pitx2 isoform present in Sol8 myoblasts. Overexpression of Pitx2c in Sol8 myoblasts inhibited myocyte differentiation and myotube formation. Furthermore, Sol8 cells overexpressing Pitx2c maintained high proliferative capacity and a significant up-regulation of the cell cycle genes cyclin D1, cyclin D2, and c-myc. Gene expression analysis for Pax3 and the s MyoD and myogenin showed that Pitx2c-overexpression caused Sol8 cells to remain as myoblasts, in an undifferentiated myogenic state. Furthermore, down-regulation of the muscle-specific genes sTnI and MyHC3 demonstrated that Sol8-overexpressing Pitx2c myoblasts failed to reach terminal differentiation. This study sheds light on previously unknown functions of the Pitx2c isoform in balancing proliferation vs. differentiation in a myogenic cell line.     

PAX6 mutations reviewed

Mutations inPAX6 are responsible for human aniridia and have also been found in patients with Peter's anomaly, with congenital cataracts, with autosomal dominant keratitis, and with isolated foveal hypoplasia. No locus other than chromosome 11p13 has been implicated in aniridia, and PAX6 is clearly the major, if not only, gene responsible. Twenty-eight percent of identified PAX6 mutations are C–T changes at CpG dinucleotides, 20% are splicing errors, and more than 30% are deletion or insertion events. There is a noticeably elevated level of mutation in the paired domain compared with the rest of the gene. Increased mutation in the homeodomain is accounted for by the hypermutable CpG dinucleotide in codon 240. Very nearly all mutations appear to cause loss of function of the mutant allele, and more than 80% of exonic substitutions result in nonsense codons. In a gene with such extraordinarily high sequence conservation throughout evolution, there are presumed undiscovered missense mutations, these are hypothesized to exist in as-yet unidentified phenotypes. Hum Mutat 11:93–108, 1998. © 1998 Wiley-Liss, Inc.     

Mutations in the human RAX homeobox gene in a patient with anophthalmia and sclerocornea

Anophthalmia and microphthalmia are among the most common ocular birth defects and a significant cause of congenital blindness. The etiology of anophthalmia and microphthalmia is diverse, with multiple genetic mutations associated with each of these conditions, along with potential environmental causes. Based on findings that mutations in the Rx/Rax homeobox genes in mice and fish lead to defects in retinal development and result in animal models of anophthalmia, we screened 75 individuals with anophthalmia and/or microphthalmia for mutations in the human RAX gene. We identified a single proband from this population who is a compound heterozygote for mutations in the RAX gene. This individual carries a truncated allele (Q147X) and a missense mutation (R192Q), both within the DNA-binding homeodomain of the RAX protein, and we have characterized the biochemical properties of these mutations in vitro. Parents and grandparents of the proband were found to be carriers without visible ocular defects, consistent with an autosomal recessive inheritance pattern. This is the first report of genetic mutations in the human RAX gene.