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Mutation in PITX2 is associated with ring dermoid of the cornea

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K Xia
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L Wu
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Xionghao Liu at Central South University
Xionghao Liu
X Xi
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X Xi
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ONLINE MUTATION REPORT
Mutation in PITX2 is associated with ring dermoid of the
cornea
K Xia, L Wu, X Liu, X Xi, D Liang, D Zheng, F Cai, Q Pan, Z Long, H Dai, Z Hu, B Tang, Z Zhang,
J Xia
...............................................................................................................................
J Med Genet 2004;41:e129 (http://www.jmedgenet.com/cgi/content/full/41/12/e129). doi: 10.1136/jmg.2004.022434
R
ing dermoid of the cornea (RDC, MIM180550) is an
autosomal dominantly inherited syndrome charac-
terised by bilateral annular limbal dermoids with
corneal and conjunctival extension. The genetic basis of
RDC is unknown. We report linkage of chromosome 4q24-
q26 to RDC and identification of a missense mutation in
PITX2 in 17 disease affected individuals but not in eight
genetically related normal individuals in a large Chinese
family.
METHODS
A large Chinese family with 17 individuals affected by the
RDC was identified (figs 1 and 2). All patients were
diagnosed by the same physician (XHX). Informed written
consent for blood sample collection was obtained from all
participants.
Linkage analysis and genotyping were done essentially as
previously described.
1
Genome-wide screening was carried
out with 382 microsatellite markers covering all autosomal
chromosomes, with an average interval of 10 cM (ABI
PRISM
TM
linkage mapping set, version 2.0). Fine mapping
was accomplished using fluorescent labelled primers from
the Marshfield database. Alleles were analysed by GeneScan
analysis, version 3.0 software, and Genotyper, version 2.1
software (ABI PRISM
TM
linkage analysis and haplotype
construction). Two point linkage analysis was conducted
using the MLINK program of the Linkage 5.1 package
(Rockefeller University, New York, USA). The disease allele
frequency was set at 0.0001 with the recombination fraction
(h) in male and female subjects considered equal. The
penetrance frequency of the disease was assumed to be
0.99 with autosomal dominance. The most likely haplotype
was constructed by the Cyrillic program (Electrotechnical
Laboratory, Tokyo, Japan).
For mutation analysis, all exons and intron–exon bound-
aries of selected genes were amplified by polymerase chain
reaction (PCR) using genomic DNA of the proband as
template and primers designed according to the genomic
sequences obtained from Genbank. Sequencing of the PCR
products was automated (ABI3100 sequencer).
RESULTS
In this large Chinese family, 21 of 36 genetically linked
individuals were affected by RDC.
2
Patients showed yellow-
white tumour-like apophyses (2–3 mm high and 3–5 mm
wide) on the corneal border of both eyes (fig 1). The
apophyses were clinically detectable at birth and progres-
sively impaired the patients’ vision with aging. Some affected
individuals also had glaucoma (II-2, III-4, and IV-3),
unilateral cataracts (IV-3), or involuntary oscillation of the
eyes (IV-3). The only clinical manifestation in the affected
individuals were in the eyes. Consistent with previously
reported autosomal dominantly inherited patterns of RDC,
affected cases were found in both male and female
descendants of each of four generations.
The linkage analysis was carried out with samples collected
from 17 affected individuals, eight genetically related normal
Key points
N
The ring dermoid of the cornea (RDC, MIM180550) is
an autosomal dominantly inherited syndrome charac-
terised by bilateral annular limbal dermoids with
corneal and conjunctival extension.
N
We report linkage of a 15 cM interval on chromosome
4q24-q26 to RDC. A missense mutation in PITX2 was
found in 17 disease affected individuals but not in eight
genetically related normal individuals in a large
Chinese family, and in 157 normal unrelated indivi-
duals.
N
Given that PITX2 functions in eye development, these
findings suggest mutations in PITX2 as a potential
cause of RDC.
Abbreviations: RDC, ring dermoid of the cornea
Figure 1 Eyes affected by ring dermoid of the cornea (RDC) in two
patients, II-2 (upper panel) and IV-3 (lower panel). Yellow-white tumour-
like apophyses are visible on the corneal border of both eyes. The
apophyses are diffuse in the superficial layer of the cornea and
conjunctiva. The corneal border is not clear and the diameter of the
transparent region of cornea is diminished to about 7–8 mm. The
upward shift of the right pupil of IV-3 is caused by cataract resection (the
affected individual, IV-3, also has bilateral glaucoma and congenital
cataracts in the right eye).
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siblings, and seven genetically unrelated family members.
Results showed a maximum two point lod score of 3.91
(h = 0), 2.33 (h = 0), and 1.87 (h = 1) for D4S1572, D4S406,
and D4S402, respectively. The results establish a linkage of
RDC to chromosome 4q. Fine mapping using 12 micro-
satellite markers around D4S1572 and D4S406 identified a
maximum two point lod score of 6.72 ( h = 0) for D4S2989.
The lod scores for the neighbouring markers D4S2945 and
D4S161 were 5.04 and 5.01 (h = 0), respectively (table 1).
Haplotype analysis for 14 markers on chromosome 4q
showed that all affected family members were carriers of
the risk haplotype (fig 2). Recombination between D4S1560
and D4S2966 was detected in affected individuals II-9 and
III-15. Another recombination between D4S1613 and
D4S1522 was found in affected individuals III-8 and IV-6.
Most normal members of the family who were examined did
not carry the haplotype. However, the genetically related
normal individual II-11 carried a partial haplotype with a
recombination between D4S1572 and D4S1570, suggesting
that the candidate risk gene is located distal to D4S1572.
Moreover, a genetically related normal individual, IV-2,
carried the haplotype with a recombination between
D4S1613 and D4S1522, where a recombination was detected
in III-8 and IV-6. The results indicate that the candidate risk
gene is located at the proximal side of D4S1522 (fig 2).
Together, the haplotype between D4S1572 and D4S1522 co-
segregates with the disease in this family. Thus a linkage of
the RDC locus to a 15 cM interval between D4S1572 and
D4S1522 was established.
The genomic interval between D4S1572 and D4S1522
contains 65 known genes and 56 reference genes. Three
potential candidate genes for RDC were chosen for further
mutation examination, based on both their tissue specific
expression and their roles in regulating cell proliferation,
differentiation, and migration that may play an important
part in RDC pathogenesis.
2
These include IDAX (NM
025212),
3
TM4SF9 (NM005723),
4
and PITX2 (NM 153427).
5
Mutation analysis showed no mutations in IDAX or TM4SF9
Figure 2 Recombination analysis of the family with ring dermoid of the
cornea (RDC). Pedigree of the family affected by RDC and haplotype
analysis for 14 markers on 4q22-q26. Markers (from top to bottom) are
centromere-D4S1560-D4S2966-D4S1572-D4S1570-D4S1564-
D4S2945-D4S2989-D4S1616-D4S406-D4S193-D4S1613-D4S1522-
D4S1612-D4S427-telomere. The haplotype co-segregating with the
disorder is boxed.
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of the proband. A heterozygous mutation of guanine to
adenine (185GRA) was detected in PITX2 of the proband
(fig 3). Further sequence analysis showed a perfect segrega-
tion of this mutation with the disease in all 17 affected
individuals in the family. Interestingly, affected individual
III-12 showed a homozygous mutation. The mother of III-12
is an affected person harbouring a heterozygous G185A
mutation, while the father is normal, with no mutation at
nucleotide 185. Genetic analysis confirms that the father is
the biological father (not shown). It is likely that III-12
carries a mutation inherited from the mother and a de novo
mutation at the same position. No mutation of PITX2 was
detected in eight genetically related normal individuals,
seven genetically unrelated individuals in the family, and
150 ethnically appropriate normal controls. The results
indicate that the sequence change observed is not a common
polymorphism.
COMMENT
PITX2, a downstream target of wnt/b-catenin pathway,
encodes a homeodomain transcription factor required for
normal development of multiple organs, including eye, heart,
and pituitary.
6–13
Mutations in PITX2 are associated with
multiple dominantly inherited diseases related to malfunc-
tion of the eyes, including Riger syndrome,
5
iridogoniodys-
genesis,
14
iris hypoplasia,
15
and Peter’s anomaly.
16
This study
suggests that mutation in PITX2 is linked to another eye
disease. The PITX2 G185A mutation found in RDC patients is
a novel disease associated mutation resulting in a substitu-
tion of arginine by histidine at amino acid 62 (R62H) located
in the conserved DNA binding homeodomain (fig 4). The
mutation probably results in changes in its transcriptional
activity, as with other disease associated mutations identified
in this gene.
17–21
Identification of this novel mutation in PITX2
may reveal the molecular mechanism underlying the RDC
pathology.
ACKNOWLEDGEMENTS
This study was supported by Chinese 973 projects (G1998051002 and
2001CB510302), 863 projects (2002BA711A07–08,03, Z19–02–02–02,
2001AA227011, and 2002BA711A08), the Chinese National Natural
Science Foundation (39980018, 30070410, 30270735, and 30340078),
the Cheung Kong Scholars Programme, and the Life Science Research
Foundation of Hunan Province.
Authors’ affiliations
.....................
K Xia, L Wu, X Liu, D Liang, D Zheng, F Cai, Q Pan, Z Long, H Dai,
Z Hu, Z Zhang, J Xia, National Laboratory of Medical Genetics of
China, Central South University, Changsha, Hunan, China
X Xi, Xiangya 2nd Hospital, Central South University
B Tang, Xiangya Hospital, Central South University
Conflicts of interest: none declared
Correspondence to: Dr K Xia, National Laboratory of Medical Genetics
of China, Central South University, Changsha, Hunan, China; nlmglcy@
xysm.net
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Figure 3 Partial sequence chromatographs of PITX2. Normal PITX2
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Online mutation report 3of4
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Table 1 Two point LOD scores between the disease gene and 14 markers of chromosome 4q22-q26
Locus Genetic distance
LOD SCORE AT h = *
Z
max
h
max
0.0 0.1 0.2 0.3 0.4
D4S1560 104.75 22.50 1.25 1.17 0.86 0.45 1.25 0.1
D4S2966 106.89 3.41 2.87 2.26 1.57 0.81 3.41 0
D4S1572 107.95 3.91 3.41 2.72 1.88 0.93 3.91 0
D4S1570 109.02 3.58 2.98 2.31 1.56 0.73 3.58 0
D4S1564 112.62 6.07 5.04 3.90 2.66 1.30 6.07 0
D4S2945 116.37 5.04 4.21 3.30 2.27 1.11 5.04 0
D4S2989 117.06 6.72 5.64 4.44 3.09 1.55 6.72 0
D4S1616 117.06 5.01 4.33 3.47 2.43 1.21 5.01 0
D4S406 117.06 2.33 1.91 1.45 0.95 0.39 2.33 0
D4S193 117.06 6.07 5.08 3.97 2.74 1.35 6.07 0
D4S1613 121.61 6.63 5.57 4.38 3.05 1.53 6.63 0.1
D4S1522 123.13 0.64 3.93 3.31 2.38 1.21 3.93 0.1
D4S1612 124.45 23.79 1.85 1.59 1.14 0.60 1.85 0.1
D4S402 124.45 21.97 1.87 1.63 1.19 0.63 1.87 0.1
D4S427 124.45 23.69 1.40 1.19 0.76 0.32 1.40 0.1
*LOD scores were calculated under an autosomal dominant mode of inheritance and a penetrance of 100%.
Sex averaged genetic distance from the next marker in centimorgans according to the Genethon human genetic linkage map (1996).
Figure 4 Sequence comparison of the homeodomain of human PITX2 and several proteins related to the homeodomain containing protein bicoid. The
missense mutation R68H in a conserved amino acid detected in patients with ring dermoid of the cornea is indicated.
4of4 Online mutation report
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... Missing and misplaced teeth [90]; Esophageal Squamous Cell Carcinoma (ESCC) [115] Visual system Axenfeld Rieger Syndrome type 1 (ARS 180,400) [90]; anterior segment dysgenesis 4 (ASSGD4 137,600); ring dermoid of cornea (180,550) [93]; iridogoniodysgenesis syndrome (IGDS); Peters ...
... Several other mutations of PITX2 have been reported to be associated with different ophthalmic conditions. A missense PITX2 mutation was found in patients diagnosed with ring dermoid of the cornea [93]. Peters' anomaly (combination of corneal opacity, defects in the posterior layers of the cornea, and lenticulo-corneal and/or irido-corneal adhesions) and persistent hyperplastic primary vitreous is known to be associated with a missense mutation of C to A at 649 bp in PITX2 coding region [94]. ...
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Homeobox genes encode sequence-specific transcription factors (SSTFs) that recognize specific DNA sequences and regulate organogenesis in all eukaryotes. They are essential in specifying spatial and temporal cell identity and as a result, their mutations often cause severe developmental defects. Pitx genes belong to the PRD class of the highly evolutionary conserved homeobox genes in all animals. Vertebrates possess three Pitx paralogs, Pitx1, Pitx2, and Pitx3 while non-vertebrates have only one Pitx gene. The ancient role of regulating left–right (LR) asymmetry is conserved while new functions emerge to afford more complex body plan and functionalities. In mouse, Pitx1 regulates hindlimb tissue patterning and pituitary development. Pitx2 is essential for the development of the oral cavity and abdominal wall while regulates the formation and symmetry of other organs including pituitary, heart, gut, lung among others by controlling growth control genes upon activation of the Wnt/ß-catenin signaling pathway. Pitx3 is essential for lens development and migration and survival of the dopaminergic neurons of the substantia nigra. Pitx gene mutations are linked to various congenital defects and cancers in humans. Pitx gene family has the potential to offer a new approach in regenerative medicine and aid in identifying new drug targets.
View
... (ring dermoid of the cornea, RDC) hypolasia, IH)Axenfeld-Rieger(Axenfeld-Rieger syndrome, ARS)PITX2 [1,8~9] . 3 PITX2 : R62H, RDC [8] ; R84W, IH, T68P, ARS. , 3 , . ...
... , ; 3 1 , [8] . R84W 5 , 15 ...
View
... Several reports also implicate PITX2 in heart phenotypes including atrial fibrillation (AF) (7)(8)(9)(10)(11). In addition, one paper describes a family with a rare phenotype of ring dermoid of the cornea [MIM180550] carrying a pathogenic variant in PITX2 (12). ...
... Mice with deficiency of Dkk2 (an extracellular antagonist of canonical Wnt-b-catenin signaling and a previously identified downstream target of Pitx2) show corneal abnormalities, including a complete fate shift from corneal epithelium to stratified epidermis in some animals; the shift is attributed to aberrant regulation of localized Wnt signaling throughout the ocular mesenchyme and limbus (21,59). In humans, one report connected a missense mutation in PITX2, p.(Arg62His), with ring dermoid of the cornea [MIM180550] in a large Chinese pedigree (12,60,61). Genetic studies in other human pedigrees affected with corneal dermoid and related phenotypes (62) may provide additional information about possible other PITX2 mutations in these conditions. ...
PITX2 deficiency and associated human disease: Insights from the zebrafish model
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The PITX2 (paired-like homeodomain 2) gene encodes a bicoid-like homeodomain transcription factor linked with several human disorders. The main associated congenital phenotype is Axenfeld-Rieger syndrome, type 1 (ARS), an autosomal dominant condition characterized by variable defects in the anterior segment of the eye, an increased risk of glaucoma, craniofacial dysmorphism and dental and umbilical anomalies; in addition to this, one report implicated PITX2 in ring dermoid of the cornea and a few others described cardiac phenotypes. We report three novel PITX2 mutations- c.271C>T, p.(Arg91Trp); c.259T>C, p.(Phe87Leu); and c.356delA, p.(Gln119Argfs*36)- identified in independent families with typical ARS characteristics and some unusual features such as corneal guttata, Wolf-Parkinson-White syndrome, and hyperextensibility. To gain further insight into the diverse roles of PITX2/pitx2 in vertebrate development, we generated various genetic lesions in the pitx2 gene via TALEN-mediated genome editing. Affected homozygous zebrafish demonstrated congenital defects consistent with the range of PITX2-associated human phenotypes: abnormal development of the cornea, iris, and iridocorneal angle; corneal dermoids; and craniofacial dysmorphism. In addition, via comparison of pitx2M64*and wild-type embryonic ocular transcriptomes we defined molecular changes associated with pitx2 deficiency, thereby implicating processes potentially underlying disease pathology. This analysis identified numerous affected factors including several members of the Wnt pathway and collagen type I and V gene families. These data further support the link between PITX2 and the WNT pathway and suggest a new role in regulation of collagen gene expression during development.
View
... E53D is also juxtaposed to F58, the residue that is known to contribute to the hydrophobic network and conformation of the homeodomain (Vieira et al. 2006). R62H (G185A), present in the highly conserved region of PITX2 was identified in 17 members of a Chinese family affected by ring dermoid of the cornea (Xia et al. 2004). R62H has also been previously linked with ARS (Amendt et al. 2000). ...
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Paired homologous domain transcription factor 2 (PITX2) is critically involved in ocular and cardiac development. Mutations in PITX2 are consistently reported in association with Axenfeld-Rieger syndrome, an autosomal dominant genetic disorder and atrial fibrillation, a common cardiac arrhythmia. In this study, we have mined missense mutations in PITX2 gene from NCBI-dbSNP and Ensembl databases, evaluated the pathogenicity of the missense variants in the homeodomain and C-terminal region using five in silico prediction tools SIFT, PolyPhen2, GERP, Mutation Assessor and CADD. Fifteen homeodomain mutations G42V, G42R, R45W, S49Y, R53W, E53D, E55V, R62H, P65S, R69H, G75R, R84G, R86K, R87W, R91P were found to be highly pathogenic by both SIFT, PolyPhen2 were further functionally characterized using I-Mutant 2.0, Consurf, MutPred and Project Hope. The findings of the study can be used for prioritizing mutations in the context of genetic studies.
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... T he homeodomain-containing transcription factor PITX2 plays a role in the regulation of ocular development, and mutations in the gene lead to several anterior segment dysgeneses, such as Axenfeld-Rieger anomaly, Peters anomaly, iris hypoplasia, and iridogoniodysgenesis. 1,2 A heterozygous mutation in the PITX2 gene on chromosome 4q25 has been also associated with a phenotype described as corneal ring dermoid by previous authors. 3,4 Corneal plana is a rare hereditary disease that can present as autosomal dominant or recessive disease (CNA1 and CNA2, respectively), both related to mutations in chromosome 12. 5 Both forms present with reduced corneal curvature, indistinct border of corneal limbus, and arcus lipoides at an early age, but while CNA1 is mild and not necessarily associated with other than corneal abnormalities, CNA2 is characterized by reduced visual activity, extreme hypermetropia, sometimes central stromal clouding, and iridocorneal adhesions. 5, 6 We herein present a patient with corneal plana-like phenotype and a pathogenic variant of PITX2, which was previously associated with corneal ring dermoid phenotype. ...
Corneal Plana-Like Phenotype With Peripheral Scleralization Associated With a Pathogenic Variant of PITX2: A Case Report
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Purpose: The aim of this study was to report a case of corneal plana-like phenotype with bilateral peripheral scleralization associated with a PITX2 pathogenic variant. Methods: Clinical findings were obtained by ophthalmologic examination. Molecular diagnosis was performed by whole-exome sequencing in the patient and his parents. Results: A 12-month-old male patient present with bilateral peripheral corneal scleralization, corneal plana-like phenotype, and iris hypoplasia. The genetic analysis revealed a de novo PITX2 pathogenic variant (c.323G>A, p.R108H). Conclusions: PITX2 c.323G>A (p.R108H) can be associated with a unique corneal plana-like phenotype with peripheral scleralization, and thus, PITX2 should be targeted in genetic testing of this specific phenotype.
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... Thus, the Pitx2 gene plays a crucial role in organ development by controlling cell proliferation and cycle. Mutation and abnormality of PITX2 are associated with disease and physiological disorders, like prostate cancer (Jiang et al. 2019), non-syndromic orodental anomalies (Intarak et al. 2018), breast cancer (Aubele et al. 2017), iris hypoplasia (Kimura et al. 2014), ring dermoid of the cornea (Xia 2004), and Axenfeld-Rieger syndrome (Meyer-Marcotty et al. 2008). PITX2 protein is expressed in PGCs, spermatogonia, and primary spermatocytes during embryonic and postnatal development in rat gonads (Nandi et al. 2011). ...
Pitx2 suppression at meiotic stages associates with seasonal inhibition of testis development in Rattus norvegicus caraco
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The bicoid‐related transcription factor 2 (Pitx2) plays a crucial role in the development of many organs and tissues by affecting the mitotic cell cycle. Postnatal testis development is related to mitosis and meiosis in multiple cell types, but the role of Pitx2 gene in seasonal inhibition of testicular development remains unknown in rodents. We analyzed PITX2 protein and Pitx2 mRNA expression features using both laboratory and wild male Rattus norvegicus caraco. In postnatal testicle of laboratory colony, we found that PITX2 was expressed in Leydig cells, pachytene spermatocytes, round spermatids, and elongating spermatids rather than spermatogonia and leptotene/zygotene spermatocytes. Pitx2b expression significantly increased along with the occurrence of pachytene spermatocytes and round spermatids, while decreased along with the processes of elongated spermatids. In wild male rats with similar testes weight, a significantly suppressed Pitx2b expression occurred with an active meiotic stage in the inhibited testes in autumn and winter, compared with the normally developing testes in spring and summer. These results indicate that Pitx2b expression suppression plays a crucial role in the seasonal inhibition of testis development. This article is protected by copyright. All rights reserved
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... Mutations in the homeobox transcription factor2 gene have been linked to Axenfeld Rieger syndrome [15][16][17][18][19], distinct ocular defects [20][21][22] and congenital heart defects [23]. Given the complexity of the ocular, facial and cardiac phenotypes revealed by PITX2c lossof-function in mice and man, it was suggested that PITX2 mutations could be underlying CHARGE syndrome but direct sequencing of 29 affected individuals did not reveal any mutation [24]. ...
Novel PITX2 Homeodomain-Contained Mutations from ATRIAL Fibrillation Patients Deteriorate Calcium Homeostasis
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Full-text available
  • May 2021
Atrial fibrillation (AF) is the most common cardiac arrhythmia in the human population, with an estimated incidence of 1–2% in young adults but increasing to more than 10% in 80+ years patients. Pituitary Homeobox 2, Paired Like Homeodomain 2 (PITX2c) loss-of-function in mice revealed that this homeodomain (HD)-containing transcription factor plays a pivotal role in atrial electrophysiology and calcium homeostasis and point to PITX2 as a candidate gene for AF. To address this issue, we recruited 31 AF patients for genetic analyses of both the known risk alleles and PITX2c open reading frame (ORF) re-sequencing. We found two-point mutations in the homedomain of PITX2 and three other variants in the 5’untranslated region. A 65 years old male patient without 4q25 risk variants but with recurrent AF displayed two distinct HD-mutations, NM_000325.5:c.309G>C (Gln103His) and NM_000325.5:c.370G>A (Glu124Lys), which both resulted in a change within a highly conserved amino acid position. To address the functional impact of the PITX2 HD mutations, we generated plasmid constructs with mutated version of each nucleotide variant (MD4 and MD5, respectively) as well as a dominant negative control construct in which the PITX2 HD was lacking (DN). Functional analyses demonstrated PITX2c MD4 and PITX2c MD5 decreased Nppa-luciferase transactivation by 50% and 40%, respectively, similar to the PITX2c DN (50%), while Shox2 promoter repression was also impaired. Co-transactivation with other cardiac-enriched co-factors, such as Gata4 and Nkx2.5, was similarly impaired, further supporting the pivotal role of these mutations for correct PITX2c function. Furthermore, when expressed in HL1 cardiomyocyte cultures, the PITX2 mutants impaired endogenous expression of calcium regulatory proteins and induced alterations in sarcoplasmic reticulum (SR) calcium accumulation. This favored alternating and irregular calcium transient amplitudes, causing deterioration of the beat-to-beat stability upon elevation of the stimulation frequency. Overall this data demonstrate that these novel PITX2c HD-mutations might be causative of atrial fibrillation in the carrier.
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... [Arg62His]), which was previously found in a family reported to have "ring dermoid" of the cornea. Notably the phenotypic images in this family also showed peripheral sclerocornea, 26 very similar to our patient (Fig. 2g, 4c, Table 1). This highlights peripheral sclerocornea as a new phenotypic link to variation in PITX2. ...
Revealing hidden genetic diagnoses in the ocular anterior segment disorders
Article
Full-text available
  • Jun 2020
Ocular anterior segment disorders (ASDs) are clinically and genetically heterogeneous, and genetic diagnosis often remains elusive. In this study, we demonstrate the value of a combined analysis protocol using phenotypic, genomic, and pedigree structure data to achieve a genetic conclusion. We utilized a combination of chromosome microarray, exome sequencing, and genome sequencing with structural variant and trio analysis to investigate a cohort of 41 predominantly sporadic cases. We identified likely causative variants in 54% (22/41) of cases, including 51% (19/37) of sporadic cases and 75% (3/4) of cases initially referred as familial ASD. Two-thirds of sporadic cases were found to have heterozygous variants, which in most cases were de novo. Approximately one-third (7/22) of genetic diagnoses were found in rarely reported or recently identified ASD genes including PXDN, GJA8, COL4A1, ITPR1, CPAMD8, as well as the new phenotypic association of Axenfeld–Rieger anomaly with a homozygous ADAMTS17 variant. The remainder of the variants were in key ASD genes including FOXC1, PITX2, CYP1B1, FOXE3, and PAX6. We demonstrate the benefit of detailed phenotypic, genomic, variant, and segregation analysis to uncover some of the previously “hidden” heritable answers in several rarely reported and newly identified ocular ASD-related disease genes.
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... ARS is a full penetrant, but clinically and genetically heterogeneous disorder characterized by developmental anomalies involving both ocular and non-ocular structures [6]. To date, 87 mutations within the PITX2 gene have been identified including deletions, insertions, splice-site mutations, and coding region frameshift, nonsense and missense mutations [7][8][9][10][11][12][13]. ...
Accurate prediction of functional, structural, and stability changes in PITX2 mutations using in silico bioinformatics algorithms
Article
Full-text available
Mutations in PITX2 have been implicated in several genetic disorders, particularly Axenfeld-Rieger syndrome. In order to determine the most reliable bioinformatics tools to assess the likely pathogenicity of PITX2 variants, the results of bioinformatics predictions were compared to the impact of variants on PITX2 structure and function. The MutPred, Provean, and PMUT bioinformatic tools were found to have the highest performance in predicting the pathogenicity effects of all 18 characterized missense variants in PITX2, all with sensitivity and specificity >93%. Applying these three programs to assess the likely pathogenicity of 13 previously uncharacterized PITX2 missense variants predicted 12/13 variants as deleterious, except A30V which was predicted as benign variant for all programs. Molecular modeling of the PITX2 homoedomain predicts that of the 31 known PITX2 variants, L54Q, F58L, V83F, V83L, W86C, W86S, and R91P alter PITX2’s structure. In contrast, the remaining 24 variants are not predicted to change PITX2’s structure. The results of molecular modeling, performed on all the PITX2 missense mutations located in the homeodomain, were compared with the findings of eight protein stability programs. CUPSAT was found to be the most reliable in predicting the effect of missense mutations on PITX2 stability. Our results showed that for PITX2, and likely other members of this homeodomain transcription factor family, MutPred, Provean, PMUT, molecular modeling, and CUPSAT can reliably be used to predict PITX2 missense variants pathogenicity.
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Cloning and characterization of a novel bicoid-related homeobox transcription factor gene, RIEG, involved in Rieger syndrome
Article
Full-text available
  • Jan 1997
Rieger syndrome (RIEG) is an autosomal-dominant human disorder that includes anomalies of the anterior chamber of the eye, dental hypoplasia and a protuberant umbilicus. We report the human cDNA and genomic characterization of a new homeobox gene, RIEG, causing this disorder. Six mutations in RIEG were found in individuals with the disorder. The cDNA sequence of Rieg, the murine homologue of RIEG, has also been isolated and shows strong homology with the human sequence. In mouse embryos Rieg mRNA localized in the periocular mesenchyme, maxillary and mandibular epithelia, and umbilicus, all consistent with RIEG abnormalities. The gene is also expressed in Rathke's pouch, vitelline vessels and the limb mesenchyme. RIEG characterization provides opportunities for understanding ocular, dental and umbilical development and the pleiotropic interactions of pituitary and limb morphogenesis.
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Mutation in the RIEG1 Gene in Patients with Iridogoniodysgenesis Syndrome
Article
Full-text available
  • Aug 1998
Axenfeld-Rieger syndrome (ARS) and iridogoniodysgenesis syndrome (IGDS) are clinically related autosomal dominant disorders which affect the anterior segment of the eye as well as non-ocular structures. ARS patients present with iris hypoplasia, a prominent Schwalbe line, adhesions between the iris stroma and the iridocorneal angle and increased intraocular pressure. IGDS is characterized by iris hypoplasia, goniodysgenesis and increased intraocular pressure. Each syndrome also presents with non-ocular features including maxillary hypoplasia, micro and anodontia, redundant periumbilical skin, hypospadius (in males), and each has been genetically linked to chromosome 4q25. RIEG1 , the gene responsible for the 4q25 ARS phenotype, recently has been cloned. RIEG1 encodes a novel member of the bicoid class of homeobox proteins known to be active as transcription factors. Mutational analysis has previously detected several mutations in this gene in ARS individuals. We have now detected a mutation in RIEG1 which segregates with the disease phenotype in a family with IGDS. This mutation is a G-->A transition altering an arginine residue to a histidine in a highly conserved location in the second helix of the homeobox of RIEG1. This mutation indicates that IGDS and ARS are allelic variants of the same disorder. This wide variability in clinical consequences of mutations at the RIEG1 4q25 locus implicates the RIEG gene broadly in ocular and craniofacial disorders.
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A mutation in the RIEG1 gene associated with Peters’ anomaly
Article
Full-text available
  • Mar 1999
Mutations within the RIEG1 homeobox gene on chromosome 4q25 have previously been reported in association with Rieger syndrome. We report a 3' splice site mutation within the 3rd intron of the RIEG1 gene which is associated with unilateral Peters' anomaly. The mutation is a single base substition of A to T at the invariant -2 site of the 3' splice site. Peters' anomaly, which is characterised by ocular anterior segment dysgenesis and central corneal opacification, is distinct from Rieger anomaly. This is the first description of a RIEG1 mutation associated with Peters' anomaly.
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Dosage requirement of PITX2 for development of multiple organs
Article
Full-text available
  • Nov 1999
Pitx2 is a homeodomain transcription factor that is mutated in Rieger syndrome, a haploinsufficiency disorder affecting eyes and teeth. Pitx2 also has a postulated role in left-right axis determination. We assessed the requirements for Pitx2 directly by generating hypomorphic and null alleles. Heterozygotes for either allele have eye abnormalities consistent with Rieger syndrome. The ventral body wall fails to close in embryos homozygous for the null allele, leaving the heart and abdominal organs externalized and the body axis contorted. In homozygotes for either allele, the heart tube undergoes normal, rightward looping and the stomach is positioned normally. In contrast, homozygotes for both alleles exhibit right isomerization of the lungs. Thus, Pitx2 is required for left-right asymmetry of the lungs but not other organs. Homozygotes for either allele exhibit septal and valve defects, and null homozygotes have a single atrium proving that a threshold level of Pitx2 is required for normal heart development. Null homozygotes exhibit arrest of pituitary gland development at the committed Rathke pouch stage and eye defects including optic nerve coloboma and absence of ocular muscles. This allelic series establishes that Pitx2 is required for the development of mulitple organs in a dosage-sensitive manner.
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Inhibition of the Wnt Signaling Pathway by Idax, a Novel Dvl-Binding Protein
Article
Full-text available
  • Feb 2001
In attempting to clarify the roles of Dvl in the Wnt signaling pathway, we identified a novel protein which binds to the PDZ domain of Dvl and named it Idax (for inhibition of the Dvl and Axin complex). Idax and Axin competed with each other for the binding to Dvl. Immunocytochemical analyses showed that Idax was localized to the same place as Dvl in cells and that expression of Axin inhibited the colocalization of Dvl and Idax. Further, Wnt-induced accumulation of β-catenin and activation of T-cell factor in mammalian cells were suppressed by expression of Idax. Expression of Idax inXenopus embryos induced ventralization with a reduction in the expression of siamois, a Wnt-inducible gene. Idax inhibited Wnt- and Dvl- but not β-catenin-induced axis duplication. It is known that Dvl is a positive regulator in the Wnt signaling pathway and that the PDZ domain is important for this activity. Therefore, these results suggest that Idax functions as a negative regulator of the Wnt signaling pathway by directly binding to the PDZ domain of Dvl.
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A molecular basis for differential developmental anomalies in Axenfeld-Rieger syndrome
Article
  • Apr 2002
  • HUM MOL GENET
Pitx2, a bicoid-like homeodomain transcription factor and Dlx2 are two transcriptional markers observed during early tooth development. PITX2 binds to bicoid and bicoid-like elements in the Dlx2 promoter and activates this promoter 30-fold in Chinese hamster ovary cells. Mutations in PITX2 associated with Axenfeld-Rieger syndrome (ARS) provided the first link of this homeodomain transcription factor to tooth development. We are investigating the molecular basis of developmental anomalies associated with human PITX2 mutations. A phenotypically less severe ARS mutant (without tooth anomalies), PITX2 R84W, has a similar DNA binding specificity compared to wild-type PITX2 and transactivates the Dlx2 promoter. This mutation is associated with iris hypoplasia (IH); in contrast a Rieger syndrome mutation, PITX2 T68P, which presents clinically with the full spectrum of developmental anomalies (including tooth anomalies), is unable to transactivate the Dlx2 promoter. Since Dlx2expression is required for tooth and craniofacial development the lack of tooth anomalies in the patient with IH may be due to the residual activity of this mutant in activating the Dlx2 promoter. We demonstrate that PITX2 phosphorylation increases PITX2 and PITX2 R84W DNA binding. The PITX2 T68P ARS mutation occurs at a protein kinase C phosphorylation site in the homeodomain. Surprisingly, phosphorylation of PITX2 T68P is increased compared to wild-type PITX2 but has little effect on its DNA binding activity. Altogether these data suggest a molecular mechanism for tooth development involving Dlx2gene expression in ARS patients.
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Ring dermoid syndrome. A new syndrome of autosomal dominantly inherited, bilateral, annual limbal dermoids with corneal and conjunctival extension
Article
  • Jul 1980
  • ARCH OPHTHALMOL-CHIC
A new hereditary syndrome of bilateral ocular dermoids is described in five patients from three generations of a single family. There were no associated extraocular anomalies. The choristomas involved the limbus for 360 degrees, extended anteriorly onto the cornea, and extended posteriorly about 5 mm within the conjunctiva for 360 degrees. Additional clinical findings of diagnostic value were conjunctival plaques of keratinization, hairs, and corneal lipid deposition. An irregular corneal astigmatism, amblyopia, and concomitant strabismus were secondary features. The unique bilateral, annular configuration of the dermoids is pathognomonic of this syndrome. Early surgical intervention is indicated to try to improve the visual prognosis and cosmesis. At surgery, the conjunctival portion of one case was easily resected in toto, but the limbal portion required partial excision. Histopathological examination confirmed the clinical diagnosis of dermoid choristoma.
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Autosomal dominant iris hypoplasia is caused by a mutation in the Rieger Syndrome (RIEG/PITX2) gene
Article
  • Feb 1998
  • AM J OPHTHALMOL
To determine whether autosomal dominant iris hypoplasia is caused by mutations in the newly described gene for Rieger syndrome (RIEG/PITX2). Mutation screening and sequence analysis was performed in a single family. A novel mutation in the RIEG/PITX2 gene was found in all affected but no unaffected individuals. This mutation would be expected to result in an arginine to tryptophan amino acid change in the homeodomain of solurshin, the RIEG/ITX2 gene product. Autosomal dominant iris hypoplasia is caused by a defect in the same gene that is defective in many cases of Rieger syndrome.
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Sequences and expression of six new members of the tetraspanin/TM4SF family
Article
  • Aug 1998
  • Biochim Biophys Acta
Tetraspanins (or TM4SF) are expressed in a wide variety of species and regulate cell adhesion, migration, proliferation and differentiation. We have identified and sequenced six new members of the tetraspanin family, called Tspan-1-6, from human cDNA. Amino acid sequence analysis of the Tspans highlights conserved residues which may be critical to tetraspanin structure and function. The Tspans are differentially expressed in human tissues.
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Identification of a Locus for Disseminated Superficial Actinic Porokeratosis at Chromosome 12q23.2-24.1
Article
  • Jul 2000
Disseminated superficial actinic porokeratosis is an autosomal dominant cutaneous disorder characterized by many uniformly small, minimal, annular, anhidrotic, and keratotic lesions. The genetic basis for this disease is unknown. Using a genomewide search in a large Chinese family, we identified a locus at chromosome 12q23.2-24. 1 responsible for disseminated superficial actinic porokeratosis. The fine mapping study indicates that the disseminated superficial actinic porokeratosis gene is located within a 9.6 cM region between markers D12S1727 and D12S1605, with a maximum two-point LOD score of 20.53 (theta = 0.00) at D12S78. This is the first locus identified for a genetic disease where the major phenotype is porokeratosis. The study provides a map location for isolation of a gene causing disseminated superficial actinic porokeratosis.
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