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Winston W.-y. Kao - One of the best experts on this subject based on the ideXlab platform.
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Study of corneal epithelial progenitor origin and the Yap1 requirement using Keratin 12 lineage tracing transgenic mice
Scientific reports, 2016Co-Authors: Ramesh B. Kasetti, Subhash Gaddipati, Shifu Tian, Lei Xue, Winston W.-y. KaoAbstract:Key issues in corneal epithelium biology are the mechanism for corneal epithelium stem cells to maintain the corneal epithelial homeostasis and wound healing responses, and what are the regulatory molecular pathways involved. There are apparent discrepancies about the locations of the progenitor populations responsible for corneal epithelial self-renewal. We have developed a genetic mouse model to trace the corneal epithelial progenitor lineages during adult corneal epithelial homeostasis and wound healing response. Our data revealed that the early corneal epithelial progenitor cells expressing Keratin-12 originated from limbus, and gave rise to the transit amplifying cells that migrated centripetally to differentiate into corneal epithelial cells. Our results support a model that both corneal epithelial homeostasis and wound healing are mainly maintained by the activated limbal stem cells originating form limbus, but not from the corneal basal epithelial layer. In the present study, we further demonstrated the nuclear expression of transcriptional coactivator YAP1 in the limbal and corneal basal epithelial cells and its essential role for maintaining the high proliferative potential of those corneal epithelial progenitor cells in vivo.
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A Case of Solitary Nonvascularized Corneal Epithelial Dysplasia
Case reports in ophthalmological medicine, 2016Co-Authors: Tomoya Morii, Yuka Okada, Winston W.-y. Kao, Takayoshi Sumioka, Ai Izutani-kitano, Yukihisa Takada, Shizuya SaikaAbstract:Background. Epithelial dysplasia is categorized as conjunctival/corneal intraepithelial neoplasia which is a precancerous lesion. The lesion is usually developed at the limbal region and grows towards central cornea in association with neovascularization into the lesion. Here, we report a case of isolated nonvascularized corneal epithelial dysplasia surrounded by normal corneal epithelium with immune histochemical finding of ocular surface tissues cytoKeratins, for example, Keratin 13 and Keratin 12. Case Presentation. A 76-year-old man consulted us for visual disturbance with localized opacification of the corneal epithelium in his left eye. His visual acuity was 20/20 and 20/200 in his right and left eye, respectively. Slit lamp examination showed a whitish plaque-like lesion at the center of his left corneal epithelium. No vascular invasion to the lesion was found. The lesion was surgically removed and subjected to histopathological examination and diagnosed as epithelial dysplasia. Amyloidosis was excluded by direct fast scarlet 4BS (DFS) staining. Immunohistochemistry showed that the dysplastic epithelial cells express Keratin 13 and vimentin, but not Keratin 12, indicating that the neoplastic epithelial cells lacked corneal-type epithelium differentiation. Conclusions. The lesion was diagnosed as nonvascularized epithelial dysplasia of ocular surface. Etiology of the lesion is not known.
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Spatiotemporally Regulated Ablation of Klf4 in Adult Mouse Corneal Epithelial Cells Results in Altered Epithelial Cell Identity and Disrupted Homeostasis
Investigative ophthalmology & visual science, 2015Co-Authors: Emili E. Delp, Winston W.-y. Kao, Sudha Swamynathan, Shivalingappa K. SwamynathanAbstract:In previous studies, conditional disruption of Klf4 in the developing mouse ocular surface from embryonic day 10 resulted in corneal epithelial fragility, stromal edema, and loss of conjunctival goblet cells, revealing the importance of Klf4 in ocular surface maturation. Here, we use spatiotemporally regulated ablation of Klf4 to investigate its functions in maintenance of adult corneal epithelial homeostasis. Expression of Cre was induced in ternary transgenic (Klf4(LoxP/LoxP)/Krt12(rtTA/rtTA)/Tet-O-Cre) mouse corneal epithelium by doxycycline administered through intraperitoneal injections and drinking water, to generate corneal epithelium-specific deletion of Klf4 (Klf4(Δ/ΔCE)). Corneal epithelial barrier function was tested by fluorescein staining. Expression of selected Klf4-target genes was determined by quantitative PCR (QPCR), immunoblotting, and immunofluorescent staining. Klf4 was efficiently ablated within 5 days of doxycycline administration in adult Klf4(Δ/ΔCE) corneal epithelium. The Klf4(Δ/ΔCE) corneal epithelial barrier function was disrupted, and the basal cells were swollen and rounded after 15 days of doxycycline treatment. Increased numbers of cell layers and Ki67-positive proliferating cells suggested deregulated Klf4(Δ/ΔCE) corneal epithelial homeostasis. Expression of tight junction proteins ZO-1 and occludin, desmosomal Dsg and Dsp, basement membrane laminin-332, and corneal epithelial-specific Keratin-12 was decreased, while that of matrix metalloproteinase Mmp9 and noncorneal Keratin-17 increased, suggesting altered Klf4(Δ/ΔCE) corneal epithelial cell identity. Ablation of Klf4 in the adult mouse corneas resulted in the absence of characteristic corneal epithelial cell differentiation, disrupted barrier function, and squamous metaplasia, revealing that Klf4 is essential for maintenance of the adult corneal epithelial cell identity and homeostasis.
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Role of SH2-containing tyrosine phosphatase Shp2 in mouse corneal epithelial stratification.
Investigative ophthalmology & visual science, 2013Co-Authors: Lung-kun Yeh, Winston W.-y. Kao, Yujin Zhang, Hongshan Liu, Gen-sheng Feng, Chia-yang LiuAbstract:Shp2 protein tyrosine phosphatase mediates a wide variety of receptor tyrosine kinases (RTK) cell signaling. Herein, we investigate the role of Shp2 in corneal morphogenesis and homeostasis. Shp2 was conditionally knocked out (Shp2(cko)) in Krt14-rtTA;tet-O-Cre;Shp2(f/f) triple transgenic mice administrated with doxycycline (Dox) from postnatal day 1 (P1) to P10, P15, and P25, respectively. In addition, corneal epithelial debridement was performed in adult (P42) mice treated with or without Dox for 8 days (from P42-P50). Mouse eyes were then subjected to histology and immunohistochemistry. Shp2(cko) revealed impaired stratification of conjunctival and corneal epithelia during morphogenesis. Likewise, Shp2(cko) failed to restore epithelial stratification after a corneal epithelial wound in adult Shp2(cko). At the cellular level, the ratio of proliferating cell nuclear antigen (PCNA-positive)/total basal cells remained unchanged, but cells in G2/M (survivin-positive) phase was significantly increased in Shp2(cko) as compared with those in the control littermate. Interestingly, deltaN-p63 (ΔNp63) expression and the asymmetric division of the basal cells were coincidentally dampened in Shp2(cko). Transmission electron microscopic study showed that desmosome and hemidesmosome densities were reduced in the corneal epithelium of Shp2(cko). Immunohistochemistry also demonstrated that expression of E-cadherin/β-catenin junction and laminin-β1 was extensively downregulated in Shp2(cko). On the other hand, corneal epithelium lacking Shp2 remained positive for K14, Pax-6, and Keratin 12 (K12), suggesting that Shp2 was dispensable for the corneal epithelial-type differentiation. These data argued that Shp2 deficiency predominantly impacted p63-dependent cell division and cell adhesive ability, which resulted in the impairment of stratification during corneal epithelial development and wound healing.
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Monoallelic expression of Krt12 gene during corneal-type epithelium differentiation of limbal stem cells.
Investigative ophthalmology & visual science, 2010Co-Authors: Yasuhito Hayashi, Chia-yang Liu, Mindy K. Call, M. Hayashi, George F. Babcock, Yuichi Ohashi, Winston W.-y. KaoAbstract:The purpose of this study was to characterize a Krt12-Cre knock-in mouse line for corneal epithelium-specific gene ablation and to analyze the allelic selection of the Keratin 12 (Krt12) gene during corneal type-epithelium differentiation. Knock-in mice were generated by gene targeting. The authors examined the expression patterns of several reporter genes in the corneas of bitransgenic Krt12cre/+/ROSA(EGFP), Krt12Cre/+/ZEG, and Krt12Cre/+/ZAP mouse lines. Krt12 and cre recombinase (Cre) immunostaining was performed. Corneal epithelial cells from bitransgenic Krt12Cre/+/ROSA(EGFP) mice were examined by fluorescence-activated cell sorter. Mosaic and spiral expression patterns of EGFP were observed in young and adult bitransgenic Krt12Cre/+/ZEG mice, respectively. Immunostaining revealed that Cre- cells were also Krt12 negative in the corneal epithelia of Krt12Cre/-/ZAP mice. Using FACS analysis, 60% to 70% of the corneal epithelial cells from Krt12Cre/+/ROSAEGFP mice were EGFP positive, whereas 20% to 30% were negative. RT-PCR revealed that EGFP+ cells express both Krt12Cre and Krt12+ alleles, whereas EGFP- cells express only Krt12+. In the Krt12Cre/- cornea, the number of epithelial cells expressing Cre is the same as that found in Krt12Cre/Cre, which can be explained by the fragility of corneal epithelial cells that did not produce Krt12 because the Krt12Cre allele was not transcribed. These observations are consistent with the notion that clonal limbal stem cells randomly activate Krt12 alleles in the process of terminal differentiation. The authors suggest that this selection is advantageous for retaining epithelial cells expressing the Krt12+ allele and that it allows tolerance to structural mutations of Krt12.
Kohji Nishida - One of the best experts on this subject based on the ideXlab platform.
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KLF4 prevents epithelial to mesenchymal transition in human corneal epithelial cells via endogenous TGF-β2 suppression
Regenerative therapy, 2019Co-Authors: Satoko Fujimoto, Yuzuru Sasamoto, Ryuhei Hayashi, Motokazu Tsujikawa, Susumu Hara, Jodie Harrington, Kohji NishidaAbstract:Krüppel-like factor 4 (KLF4) is considered one of the Yamanaka factors, and recently, we and others have shown that KLF4 is one of the transcription factors essential for reprogramming non-human corneal epithelial cells (HCECs) into HCECs. Since epithelial to mesenchymal transition (EMT) suppression is vital for homeostasis of HCECs via regulation of transcription factors, in this study, we aimed to investigate whether KLF4 prevents EMT in HCECs and to elucidate the underlying mechanism within the canonical TGF-β signalling pathway, which is involved in corneal epithelial wound healing. HCECs were collected from cadaver donors and cultivated. We generated KLF4-knockdown (KD) HCECs using siRNA transfection and analysed morphology, gene or protein expression, and endogenous TGF-β secretion. KLF4 was overexpressed using lentiviral KLF4 expression vectors and underwent protein expression analyses after TGF-β2 treatment. KLF4-KD HCECs showed a fibroblastic morphology, downregulation of the epithelial markers, Keratin 12 and Keratin 14, and upregulation of the mesenchymal markers, fibronectin 1, vimentin, N-cadherin, and SLUG. Although E-cadherin expression remained unchanged in KLF4-KD HCECs, immunocytochemical analysis showed that E-cadherin-positive adherens junctions decreased in KLF4-KD HCECs as well as the decreased total protein levels of E-cadherin analysed by immunoblotting. Moreover, within the TGF-β canonical signalling pathway, TGF-β2 secretion by HCECs increased up to 5 folds, and several TGF-β-associated markers (TGFB1, TGFB2, TGFBR1, and TGFBR2) were significantly upregulated up to 6 folds in the KLF4-KD HCECs. SMAD2/3, the main signal transduction molecules of the TGF-β signalling pathway, were found to be localised in the nucleus of KLF4-KD HCECs. When KLF4 was overexpressed, cultivated HCECs showed upregulation of epithelial markers, Keratin 14 and E-cadherin, indicating the contributory role of KLF4 in the homeostasis of human corneal epithelium in vivo. In addition, KLF4 overexpression in HCECs resulted in decreased SMAD2 phosphorylation and altered nuclear localisation of SMAD2/3, even after TGF-β2 treatment. These results show that KLF4 prevents EMT in HCECs and suggest a novel role of KLF4 as an endogenous TGF-β2 suppressor in the human corneal epithelium, thus highlighting the potential of KLF4 to prevent EMT and subsequent corneal fibrotic scar formation by attenuating TGF-β signalling.
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PAX6 Isoforms, along with Reprogramming Factors, Differentially Regulate the Induction of Cornea-specific Genes
Scientific Reports, 2016Co-Authors: Yuzuru Sasamoto, Satoshi Kawasaki, Andrew J. Quantock, Ryuhei Hayashi, Sung-joon Park, Mihoko Saito-adachi, Yutaka Suzuki, Kenta Nakai, Motokazu Tsujikawa, Kohji NishidaAbstract:PAX6 is the key transcription factor involved in eye development in humans, but the differential functions of the two PAX6 isoforms, isoform-a and isoform-b, are largely unknown. To reveal their function in the corneal epithelium, PAX6 isoforms, along with reprogramming factors, were transduced into human non-ocular epithelial cells. Herein, we show that the two PAX6 isoforms differentially and cooperatively regulate the expression of genes specific to the structure and functions of the corneal epithelium, particularly Keratin 3 (KRT3) and Keratin 12 (KRT12). PAX6 isoform-a induced KRT3 expression by targeting its upstream region. KLF4 enhanced this induction. A combination of PAX6 isoform-b, KLF4 and OCT4 induced KRT12 expression. These new findings will contribute to furthering the understanding of the molecular basis of the corneal epithelium specific phenotype.
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Characteristics of the human ocular surface epithelium.
Progress in retinal and eye research, 2001Co-Authors: Shigeru Kinoshita, Wakako Adachi, Kohji Nishida, Andrew J. Quantock, Chie Sotozono, Norihiko Yokoi, Kousaku OkuboAbstract:An appreciation of the biological characteristics of the human ocular surface epithelium affords us a great insight into the physiology of the human ocular surface in health and disease. Here, we review five important aspects of the human ocular surface epithelium. First, we recognize the discovery of corneal epithelial stem cells, and note how the palisades of Vogt have been suggested as a clinical marker of their presence. Second, we introduce the concept of the gene expression profile of the ocular surface epithelium as arrived at using a new strategy for the systematic analysis of active genes. We also provide a summary of several genes abundantly or uniquely expressed in the human corneal epithelium, namely clusterin, Keratin 3, Keratin 12, aldehyde dehydrogenase 3 (ALDH3), troponin-I fast-twitch isoform, sig-h3, cathepsin L2 (cathepsin V), uroplakin Ib, and Ca2+-activated chloride channel. Genes related to limbal and conjunctival epithelia are also described. Third, we touch upon the genetic abnormalities thought to be involved with epithelial dysfunction in Meesmann's dystrophy, gelatinous drop-like corneal dystrophy, and the sig-h3-mutated corneal dystrophies. Fourth, we provide an update regarding the current state of knowledge of the role of cytokines, growth factors and apoptosis in relation to ocular surface homeostasis and tissue reconstruction; the main factors being epidermal growth factor (EGF), Keratinocyte growth factor (KGF), hepatocyte growth factor (HGF), transforming growth factor-s (TGF-s), and some inflammatory cytokines. Fifth, corneal epithelial barrier function and dysfunction as measured by fluorophotometry is remarked upon, with an explanation of the FL-500 fluorophotometer and its ability to detect corneal epithelial dysfunction at a subclinical level. The research described in this review has undoubtedly generated a complete understanding of corneal epithelial pathophysiology—an understanding that, directly or indirectly, has helped advance the development of new therapeutic modalities for ocular surface reconstruction.
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Isolation and chromosomal localization of a cornea-specific human Keratin 12 gene and detection of four mutations in Meesmann corneal epithelial dystrophy.
American Journal of Human Genetics, 1997Co-Authors: Kohji Nishida, Yoichi Honma, Wakako Adachi, Hisashi Hosotani, Shuji Yamamoto, Takahiro Nakamura, Atsuyoshi Dota, Satoshi Kawasaki, Andrew J. Quantock, Masaki OkadaAbstract:Summary Keratin 12 (K12) is an intermediate-filament protein expressed specifically in corneal epithelium. Recently, we isolated K12 cDNA from a human corneal epithelial cDNA library and determined its full sequence. Herein, we present the exon-intron boundary structure and chromosomal localization of human K12. In addition, we report four K12 mutations in Meesmann corneal epithelial dystrophy (MCD), an autosomal dominant disorder characterized by intraepithelial microcysts and corneal epithelial fragility in which mutations in Keratin 3 (K3) and K12 have recently been implicated. In the human K12 gene, we identified seven introns, defining eight individual exons that cover the coding sequence. Together the exons and introns span ∼6 kb of genomic DNA. Using FISH, we found that the K12 gene mapped to 17q12, where a type I Keratin cluster exists. In this study, four new K12 mutations (Arg135Gly, Arg135Ile, Tyr429Asp, and Leu140Arg) were identified in three unrelated MCD pedigrees and in one individual with MCD. All mutations were either in the highly conserved α-helix–initiation motif of rod domain 1A or in the α-helix–termination motif of rod domain 2B. These sites are essential for Keratin filament assembly, suggesting that the mutations described above may be causative for MCD. Of particular interest, one of these mutations (Tyr429Asp), detected in both affected individuals in one of our pedigrees, is the first mutation to be identified within the α-helix–termination motif in type I Keratin.
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Isolation and chromosomal localization of a cornea-specific human Keratin 12 gene and detection of four mutations in Meesmann corneal epithelial dystrophy.
American journal of human genetics, 1997Co-Authors: Kohji Nishida, Yoichi Honma, Wakako Adachi, Hisashi Hosotani, Shuji Yamamoto, Takahiro Nakamura, Atsuyoshi Dota, Satoshi Kawasaki, Andrew J. Quantock, Masaki OkadaAbstract:Keratin 12 (K12) is an intermediate-filament protein expressed specifically in corneal epithelium. Recently, we isolated K12 cDNA from a human corneal epithelial cDNA library and determined its full sequence. Herein, we present the exon-intron boundary structure and chromosomal localization of human K12. In addition, we report four K12 mutations in Meesmann corneal epithelial dystrophy (MCD), an autosomal dominant disorder characterized by intraepithelial microcysts and corneal epithelial fragility in which mutations in Keratin 3 (K3) and K12 have recently been implicated. In the human K12 gene, we identified seven introns, defining eight individual exons that cover the coding sequence. Together the exons and introns span approximately 6 kb of genomic DNA. Using FISH, we found that the K12 gene mapped to 17q12, where a type I Keratin cluster exists. In this study, four new K12 mutations (Arg135Gly, Arg135Ile, Tyr429Asp, and Leu140Arg) were identified in three unrelated MCD pedigrees and in one individual with MCD. All mutations were either in the highly conserved alpha-helix-initiation motif of rod domain 1A or in the alpha-helix-termination motif of rod domain 2B. These sites are essential for Keratin filament assembly, suggesting that the mutations described above may be causative for MCD. Of particular interest, one of these mutations (Tyr429Asp), detected in both affected individuals in one of our pedigrees, is the first mutation to be identified within the alpha-helix-termination motif in type I Keratin.
Candace W.-c. Kao - One of the best experts on this subject based on the ideXlab platform.
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Characterization of tetracycline-inducible bitransgenic Krt12rtTA/+/tet-O-LacZ mice.
Investigative ophthalmology & visual science, 2005Co-Authors: Tai-ichiro Chikama, Chia-yang Liu, Candace W.-c. Kao, Kazuto Terai, Yasuhito Hayashi, M. Hayashi, Noriko Terai, L. Wang, Teruo Nishida, Philip SanfordAbstract:To prepare binary transgenic mouse lines that overexpress reporter genes in a corneal-epithelium-specific manner when induced by doxycycline. A gene-targeting construct containing an internal ribosomal entry site-reverse tetracycline transcription activator (IRES-rtTA) cassette was inserted into the Krt12 allele (Keratin 12 gene) to produce a knock-in Krt12(rtTA/+) mouse line through gene-targeting techniques. The Krt12(rtTA/+) knock-in mice were bred with tet-O-LacZ reporter mice to obtain Krt12(rtTA/+)/tet-O-LacZ bitransgenic mice. The expression of the LacZ gene was induced in bitransgenic mice by administration of doxycycline in the drinking water and chow. Administration of doxycycline induced a 15-fold increase of beta-galactosidase activity in the cornea of adult bitransgenic mice (Krt12(rtTA/+)/tet-O-lacZ). Administration of doxycycline either to single transgenic Krt12(rtTA/+) or tet-O-LacZ mice as a control did not induce overexpression of LacZ as it did in the bitransgenic mice. The induction of beta-galactosidase enzyme activity by doxycycline in bitransgenic mice took place in 24 hours and reached a plateau by 2 days. Histochemical analysis also showed that beta-galactosidase induction was limited to the corneal epithelium of bitransgenic mice fed doxycycline. The increased beta-galactosidase activity in corneal epithelium caused by doxycycline returned to basal levels in 4 weeks after the antibiotics were omitted from the diet. A binary mouse model has been successfully established that conditionally overexpresses reporter genes in corneal epithelium. This mouse model will be useful in elucidating signaling pathways of various growth factors and cytokines and gene functions in the maintenance of homeostasis and pathogenesis in the adult mouse cornea.
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Altered KSPG expression by keratocytes following corneal injury.
Molecular vision, 2003Co-Authors: Eric C. Carlson, Paul Brannan, Ijong Wang, Chia-yang Liu, Candace W.-c. Kao, Winston W.-y. KaoAbstract:Keratocytes synthesize keratan-sulfate proteoglycans (KSPG), lumican and keratocan, to develop and maintain proper collagen interfibrillar spacing and fibril diameter characteristics of the transparent cornea. The purposes of this study are to compare the expression patterns of KSPGs and Keratin 12 (K12) respectively by corneal keratocytes and epithelial cells after three different types of injuries; partial and total epithelial debridement and alkali burn. Corneas of 8-12 week old C57Bl/6J or FVBN mice were wounded by partial epithelial (2 mm in diameter) and total epithelial debridement, and alkali burn (0.1 M NaOH, 30 s) and were allowed to heal for various periods of time, from 1 to 84 days. The corneas were then subjected to light microscopy, in situ and Northern hybridization and RT-PCR for examining the expression of K12 and KSPG in the corneal epithelium and stroma, respectively. Immunohistochemistry with anti-alpha-smooth muscle actin (alpha-SMA) was used to identify myofibroblasts in the stroma of injured cornea. In 2-3 days, partial epithelial denuded corneas were resurfaced by corneal epithelium positive for K12, and stromal edema caused by debridement disappeared. Total epithelial debridement wounded corneas were resurfaced by conjunctival epithelial cells in 2 weeks. Stromal edema in the total epithelial debridement corneas began to subside after 6 weeks. Corneal epithelial cells resurfaced alkali burned corneas within 3-5 days. In situ and Northern hybridization showed a decrease in keratocan and lumican expression at 6 weeks and increased at 12 weeks post-injury in all wound types. Alpha-SMA positive myofibroblasts in the cornea were detected via immunostaining at the time point when KSPG expression was lowest, 6 weeks post-injury. The results suggest keratocan and lumican are down-regulated during wound healing at 6 weeks and returned to higher levels at 12 weeks post-injury; implicating that the cells repopulating the injured corneal stroma regained the characteristic function of keratocytes independent of the wound types. However, complete epithelial removal results in irreversible loss of K12 expression.
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Cis-regulatory elements of the mouse Krt1.12 gene.
Molecular vision, 2002Co-Authors: Ijong Wang, Eric C. Carlson, Chia-yang Liu, Candace W.-c. Kao, Winston W.-y. KaoAbstract:Keratin 12 is a cornea epithelial cell-specific intermediate filament component. To better understand the regulatory mechanism of its expression, the cis-regulatory elements located between the transcription start site and 600 bp upstream of the Krt1.12 gene were determined. The promoter activity of reporter gene constructs containing 0.6, 0.4, and 0.2 kb of DNA 5' upstream of Krt1.12 coupled to the lac Z gene were determined in rabbit corneas using Gene Gun technology. DNA foot printing and EMSA (electrophoresis mobility shift assay) were employed to identify putative cis-regulatory elements of the Krt1.12 gene using bovine corneal epithelial cell nuclear extracts. Enzyme activity assays and histochemical analysis of beta-galactosidase from the 0.6, 0.4, and 0.2 kb K12 promoter constructs indicated that the DNA elements between -0.2 and -0.6 kb 5' of the Krt1.12 gene contain cis-regulatory elements for its corneal epithelial cell-specific expression. Foot printing and EMSA showed that the sequences between -181 to -111 and -256 to -193 upstream of the Krt1.12 gene reacted to nuclear proteins isolated from bovine corneal epithelial cells. A Genbank search revealed that these two regions were potential binding sites for many transcription factors such as AP1, c/EBP, and KLF6. Immunofluorescent staining indicated the presence of c-jun and c/EBP transcription factors in the nuclei of corneal epithelial cells. The data is consistent with the notion that the -182 to -111 and -256 to -193 fragments 5' of the Krt1.12 gene may serve as corneal epithelial cell-specific cis-regulatory elements, and the coordinated interactions of various transcription factors are required for cornea-specific expression of Krt1.12 gene.
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TGFβ2 in corneal morphogenesis during mouse embryonic development
Developmental biology, 2001Co-Authors: Mohamad Azhar, L. Philip Sanford, Robert L. Gendron, Thomas Doetschman, Shizuya Saika, Chia-yang Liu, Candace W.-c. Kao, Winston W.-y. KaoAbstract:To examine the roles of TGFbeta isoforms on corneal morphogenesis, the eyes of mice that lack TGFbetas were analyzed at different developmental stages for cell proliferation, migration and apoptosis, and for expression patterns of Keratin 12, lumican, keratocan and collagen I. Among the three Tgfb(-/-) mice, only Tgfb2(-/-) mice have abnormal ocular morphogenesis characterized by thin corneal stroma, absence of corneal endothelium, fusion of cornea to lens (a Peters'-like anomaly phenotype), and accumulation of hyaline cells in vitreous. In Tgfb2(-/-) mice, fewer keratocytes were found in stroma that has a decreased accumulation of ECM; for example, lumican, keratocan and collagen I were greatly diminished. The absence of TGFbeta2 did not compromise cell proliferation, nor enhance apoptosis. The thinner stroma resulting from decreased ECM synthesis may account for the decreased cell number in the stroma of Tgfb2 null mice. Keratin 12 expression was not altered in Tgfb2(-/-) mice, implicating normal corneal type epithelial differentiation. Delayed appearance of macrophages in ocular tissues was observed in Tgfb2(-/-) mice. Malfunctioning macrophages may account for accumulation of cell mass in vitreous of Tgfb2 null mice.
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Identification of the cornea-specific Keratin 12 promoter by in vivo particle-mediated gene transfer.
Investigative ophthalmology & visual science, 1998Co-Authors: Atsushi Shiraishi, Chia-yang Liu, Richard Converse, Candace W.-c. Kao, F. Zhou, Winston Whei-yang KaoAbstract:PURPOSE. Keratin 12 (K12) is a cornea epithelial cell-specific intermediate filament component. To provide a better understanding of its expression, it is necessary to identify and characterize the promoter of Krtl. 12 gene. METHODS. The 2.5-kb DNA 5' to Krtl.12 gene was sequenced. K12 promoter-j3-gal DNA constructs were prepared and used in vivo to transfect rabbit corneas, conjunctivas, and skin by particlemediated gene transfer (Gene Gun). In vitro, the DNA constructs were transfected into cultured T-antigen-transformed rabbit corneal epithelial (RCE-T) cells and human fibrosarcoma HT-1080 fibroblasts with lipofectamine. The promoter activity was assessed by measuring j3-gal (j3-galactosidase) activity using histochemical staining with 5-Bromo-4-chloro-3-indolyl-j3-D-galactoside and enzyme assay with o-nitrophenyl /3-D-galactopyranoside. RESULTS. There are four Pax-6 pair box binding elements found between —910 and -2000 bp 5'-flanking the transcription initiation site of the Krtl. 12 gene. None of promoter constructs can be expressed by HT-1080 cells. Cotransfection of Pax-6 cDNA with K12 promoter-j3-gal constructs containing Pax-6 elements results in a fourfold increase of /3-gal activities in RCE-T cells but not HT-1080 fibroblasts. The data of in vivo transfection in the rabbit by Gene Gun indicate that reporter gene constructs containing 0.6-kb and longer DNA fragments 5'-flanking Krtl. 12 gene are effectively expressed in corneal, but not conjunctival or epidermal epithelial cells. CONCLUSIONS. The particle-mediated gene transfer is a suitable technique for in vivo deliver)' of transgenes to corneal epithelial cells. The 2.5-kb DNA fragment 5'-flanking Krtl. 12 contains corneal epithelial cell-specific regulatory cis-DNA elements. Pax-6 is a positive transcription factor essential for Keratin 12 expression. (Invest Ophthalmol Vis Sci. 1998;39:2554-256l)
Chia-yang Liu - One of the best experts on this subject based on the ideXlab platform.
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Role of SH2-containing tyrosine phosphatase Shp2 in mouse corneal epithelial stratification.
Investigative ophthalmology & visual science, 2013Co-Authors: Lung-kun Yeh, Winston W.-y. Kao, Yujin Zhang, Hongshan Liu, Gen-sheng Feng, Chia-yang LiuAbstract:Shp2 protein tyrosine phosphatase mediates a wide variety of receptor tyrosine kinases (RTK) cell signaling. Herein, we investigate the role of Shp2 in corneal morphogenesis and homeostasis. Shp2 was conditionally knocked out (Shp2(cko)) in Krt14-rtTA;tet-O-Cre;Shp2(f/f) triple transgenic mice administrated with doxycycline (Dox) from postnatal day 1 (P1) to P10, P15, and P25, respectively. In addition, corneal epithelial debridement was performed in adult (P42) mice treated with or without Dox for 8 days (from P42-P50). Mouse eyes were then subjected to histology and immunohistochemistry. Shp2(cko) revealed impaired stratification of conjunctival and corneal epithelia during morphogenesis. Likewise, Shp2(cko) failed to restore epithelial stratification after a corneal epithelial wound in adult Shp2(cko). At the cellular level, the ratio of proliferating cell nuclear antigen (PCNA-positive)/total basal cells remained unchanged, but cells in G2/M (survivin-positive) phase was significantly increased in Shp2(cko) as compared with those in the control littermate. Interestingly, deltaN-p63 (ΔNp63) expression and the asymmetric division of the basal cells were coincidentally dampened in Shp2(cko). Transmission electron microscopic study showed that desmosome and hemidesmosome densities were reduced in the corneal epithelium of Shp2(cko). Immunohistochemistry also demonstrated that expression of E-cadherin/β-catenin junction and laminin-β1 was extensively downregulated in Shp2(cko). On the other hand, corneal epithelium lacking Shp2 remained positive for K14, Pax-6, and Keratin 12 (K12), suggesting that Shp2 was dispensable for the corneal epithelial-type differentiation. These data argued that Shp2 deficiency predominantly impacted p63-dependent cell division and cell adhesive ability, which resulted in the impairment of stratification during corneal epithelial development and wound healing.
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Monoallelic expression of Krt12 gene during corneal-type epithelium differentiation of limbal stem cells.
Investigative ophthalmology & visual science, 2010Co-Authors: Yasuhito Hayashi, Chia-yang Liu, Mindy K. Call, M. Hayashi, George F. Babcock, Yuichi Ohashi, Winston W.-y. KaoAbstract:The purpose of this study was to characterize a Krt12-Cre knock-in mouse line for corneal epithelium-specific gene ablation and to analyze the allelic selection of the Keratin 12 (Krt12) gene during corneal type-epithelium differentiation. Knock-in mice were generated by gene targeting. The authors examined the expression patterns of several reporter genes in the corneas of bitransgenic Krt12cre/+/ROSA(EGFP), Krt12Cre/+/ZEG, and Krt12Cre/+/ZAP mouse lines. Krt12 and cre recombinase (Cre) immunostaining was performed. Corneal epithelial cells from bitransgenic Krt12Cre/+/ROSA(EGFP) mice were examined by fluorescence-activated cell sorter. Mosaic and spiral expression patterns of EGFP were observed in young and adult bitransgenic Krt12Cre/+/ZEG mice, respectively. Immunostaining revealed that Cre- cells were also Krt12 negative in the corneal epithelia of Krt12Cre/-/ZAP mice. Using FACS analysis, 60% to 70% of the corneal epithelial cells from Krt12Cre/+/ROSAEGFP mice were EGFP positive, whereas 20% to 30% were negative. RT-PCR revealed that EGFP+ cells express both Krt12Cre and Krt12+ alleles, whereas EGFP- cells express only Krt12+. In the Krt12Cre/- cornea, the number of epithelial cells expressing Cre is the same as that found in Krt12Cre/Cre, which can be explained by the fragility of corneal epithelial cells that did not produce Krt12 because the Krt12Cre allele was not transcribed. These observations are consistent with the notion that clonal limbal stem cells randomly activate Krt12 alleles in the process of terminal differentiation. The authors suggest that this selection is advantageous for retaining epithelial cells expressing the Krt12+ allele and that it allows tolerance to structural mutations of Krt12.
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Eye drop delivery of nano-polymeric micelle formulated genes with cornea-specific promoters.
The journal of gene medicine, 2007Co-Authors: Yaw Chong Tong, Winston W.-y. Kao, Chia-yang Liu, Shwu Fen Chang, Chong Heng Huang, Jiahorng LiawAbstract:This study evaluates the eye drop delivery of genes with cornea-specific promoters, i.e., Keratin 12 (K12) and keratocan (Kera3.2) promoters, by non-ionic poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) (PEO-PPO-PEO) polymeric micelles (PM) to mouse and rabbit eyes, and investigates the underlying mechanisms. Three PM-formulated plasmids (pCMV-Lac Z, pK12-Lac Z and pKera3.2-Lac Z) containing the Lac Z gene for beta-galactosidase (beta-Gal) whose expression was driven by the promoter of either the cytomegalovirus early gene, the Keratin 12 gene or the keratocan gene, were characterized by critical micelle concentration (CMC), dynamic light scattering (DLS), and atomic force microscopy (AFM). Transgene expression in ocular tissue after gene delivery was analyzed by 5-bromo-4-chloro-3-indolyl-beta-D-galactoside (X-Gal) color staining, 1,2-dioxetane beta-Gal enzymatic activity measurement, and real-time polymerase chain reaction (PCR) analysis. The delivery mechanisms of plasmid-PM on mouse and rabbit corneas were evaluated by EDTA and RGD (arginine-glycine-aspartic acid) peptide. The sizes of the three plasmid-PM complexes were around 150-200 nm with unimodal distribution. Enhanced stability was found for three plasmid-PM formulations after DNase I treatment. After six doses of eye drop delivery of pK12-Lac Z-PM three times a day, beta-Gal activity was significantly increased in both mouse and rabbit corneas. Stroma-specific Lac Z expression was only found in pKera3.2-Lac Z-PM-treated animals with pretreatment by 5 mM EDTA, an opener of junctions. Lac Z gene expression in both pK12-Lac Z-PM and pKera3.2-Lac Z-PM delivery groups was decreased by RGD peptide pretreatment. Cornea epithelium- and stroma-specific gene expression could be achieved using cornea-specific promoters of Keratin 12 and keratocan genes, and the gene was delivered with PM formulation through non-invasive, eye drop in mice and rabbits. The transfection mechanism of plasmid-PM may involve endocytosis and particle size dependent paracellular transport. 2007 John Wiley & Sons, Ltd
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Characterization of tetracycline-inducible bitransgenic Krt12rtTA/+/tet-O-LacZ mice.
Investigative ophthalmology & visual science, 2005Co-Authors: Tai-ichiro Chikama, Chia-yang Liu, Candace W.-c. Kao, Kazuto Terai, Yasuhito Hayashi, M. Hayashi, Noriko Terai, L. Wang, Teruo Nishida, Philip SanfordAbstract:To prepare binary transgenic mouse lines that overexpress reporter genes in a corneal-epithelium-specific manner when induced by doxycycline. A gene-targeting construct containing an internal ribosomal entry site-reverse tetracycline transcription activator (IRES-rtTA) cassette was inserted into the Krt12 allele (Keratin 12 gene) to produce a knock-in Krt12(rtTA/+) mouse line through gene-targeting techniques. The Krt12(rtTA/+) knock-in mice were bred with tet-O-LacZ reporter mice to obtain Krt12(rtTA/+)/tet-O-LacZ bitransgenic mice. The expression of the LacZ gene was induced in bitransgenic mice by administration of doxycycline in the drinking water and chow. Administration of doxycycline induced a 15-fold increase of beta-galactosidase activity in the cornea of adult bitransgenic mice (Krt12(rtTA/+)/tet-O-lacZ). Administration of doxycycline either to single transgenic Krt12(rtTA/+) or tet-O-LacZ mice as a control did not induce overexpression of LacZ as it did in the bitransgenic mice. The induction of beta-galactosidase enzyme activity by doxycycline in bitransgenic mice took place in 24 hours and reached a plateau by 2 days. Histochemical analysis also showed that beta-galactosidase induction was limited to the corneal epithelium of bitransgenic mice fed doxycycline. The increased beta-galactosidase activity in corneal epithelium caused by doxycycline returned to basal levels in 4 weeks after the antibiotics were omitted from the diet. A binary mouse model has been successfully established that conditionally overexpresses reporter genes in corneal epithelium. This mouse model will be useful in elucidating signaling pathways of various growth factors and cytokines and gene functions in the maintenance of homeostasis and pathogenesis in the adult mouse cornea.
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Altered KSPG expression by keratocytes following corneal injury.
Molecular vision, 2003Co-Authors: Eric C. Carlson, Paul Brannan, Ijong Wang, Chia-yang Liu, Candace W.-c. Kao, Winston W.-y. KaoAbstract:Keratocytes synthesize keratan-sulfate proteoglycans (KSPG), lumican and keratocan, to develop and maintain proper collagen interfibrillar spacing and fibril diameter characteristics of the transparent cornea. The purposes of this study are to compare the expression patterns of KSPGs and Keratin 12 (K12) respectively by corneal keratocytes and epithelial cells after three different types of injuries; partial and total epithelial debridement and alkali burn. Corneas of 8-12 week old C57Bl/6J or FVBN mice were wounded by partial epithelial (2 mm in diameter) and total epithelial debridement, and alkali burn (0.1 M NaOH, 30 s) and were allowed to heal for various periods of time, from 1 to 84 days. The corneas were then subjected to light microscopy, in situ and Northern hybridization and RT-PCR for examining the expression of K12 and KSPG in the corneal epithelium and stroma, respectively. Immunohistochemistry with anti-alpha-smooth muscle actin (alpha-SMA) was used to identify myofibroblasts in the stroma of injured cornea. In 2-3 days, partial epithelial denuded corneas were resurfaced by corneal epithelium positive for K12, and stromal edema caused by debridement disappeared. Total epithelial debridement wounded corneas were resurfaced by conjunctival epithelial cells in 2 weeks. Stromal edema in the total epithelial debridement corneas began to subside after 6 weeks. Corneal epithelial cells resurfaced alkali burned corneas within 3-5 days. In situ and Northern hybridization showed a decrease in keratocan and lumican expression at 6 weeks and increased at 12 weeks post-injury in all wound types. Alpha-SMA positive myofibroblasts in the cornea were detected via immunostaining at the time point when KSPG expression was lowest, 6 weeks post-injury. The results suggest keratocan and lumican are down-regulated during wound healing at 6 weeks and returned to higher levels at 12 weeks post-injury; implicating that the cells repopulating the injured corneal stroma regained the characteristic function of keratocytes independent of the wound types. However, complete epithelial removal results in irreversible loss of K12 expression.
W.h.i. Mclean - One of the best experts on this subject based on the ideXlab platform.
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Allele-specific siRNA silencing for the common Keratin 12 founder mutation in Meesmann epithelial corneal dystrophy.
Investigative ophthalmology & visual science, 2013Co-Authors: Edwin H. A. Allen, Sarah D. Atkinson, Johnny Moore, Deena M. Leslie Pedrioli, W.h.i. Mclean, Haihui Liao, Fjd Smith, Tara MooreAbstract:PURPOSE To identify an allele-specific short interfering RNA (siRNA), against the common KRT12 mutation Arg135Thr in Meesmann epithelial corneal dystrophy (MECD) as a personalized approach to treatment. METHODS siRNAs against the K12 Arg135Thr mutation were evaluated using a dual luciferase reporter gene assay and the most potent and specific siRNAs were further screened by Western blot. Off-target effects on related Keratins were assessed and immunological stimulation of TLR3 was evaluated by RT-PCR. A modified 5' rapid amplification of cDNA ends method was used to confirm siRNA-mediated mutant knockdown. Allele discrimination was confirmed by quantitative infrared immunoblotting. RESULTS The lead siRNA, with an IC(50) of thirty picomolar, showed no Keratin off-target effects or activation of TLR3 in the concentration ranges tested. We confirmed siRNA-mediated knockdown by the presence of K12 mRNA fragments cleaved at the predicted site. A dual tag infrared immunoblot showed knockdown to be allele-specific, with 70% to 80% silencing of the mutant protein. CONCLUSIONS A potent allele-specific siRNA against the K12 Arg135Thr mutation was identified. In combination with efficient eyedrop formulation delivery, this would represent a personalized medicine approach, aimed at preventing the pathology associated with MECD and other ocular surface pathologies with dominant-negative or gain-of-function pathomechanisms.
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A novel mutation in KRT12 associated with Meesmann's epithelial corneal dystrophy.
The British journal of ophthalmology, 2002Co-Authors: Alan D. Irvine, Johnny Moore, Ole Swensson, Fjd Smith, C. M. Coleman, S J Morgan, J H Mccarthy, Graeme C.m. Black, W.h.i. McleanAbstract:The molecular basis of Meesmann's epithelial corneal dystrophy (MECD) has recently been attributed to mutations in the cornea specific Keratin genes KRT3 and KRT12. The mechanisms by which these mutations cause the Meesmann's phenotype are not clear. This study presents new data, examines clinical, histological, ultrastructural, and molecular aspects of MECD, and compares the features seen in this condition with those observed in other well studied Keratin diseases such as epidermolysis bullosa simplex. A two generation family with typical features of Meesmann's epithelial corneal dystrophy (MECD) was studied. All family members were examined under a slit lamp. Biopsy material from elective keratoplasty was studied by histopathological and ultrastructural analysis using standard techniques. Direct automated sequencing of genomic DNA was used for mutation detection, mutations were confirmed by restriction digest analysis. The abnormal corneal epithelium was acanthotic and contained numerous dyskeratotic cells and intraepithelial vesicles. By electron microscopy abnormally aggregated and clumped Keratin filament bundles were detected in basal and suprabasal Keratinocytes from the centre of the cornea. Direct sequencing of the patients' genomic DNA revealed a novel missense mutation (423T>G) in exon 1 of the cornea specific Keratin 12 (KRT12) gene. This mutation predicts the amino acid change N133K within the helix initiation motif of the K12 polypeptide. Comparative studies with well established Keratin disorders of other human epithelia underscore the pathogenic relevance of K3 and K12 gene mutations in Meesmann's epithelial corneal dystrophy. The morphological data presented here illustrate the disruptive effects of Keratin gene mutations on the integrity of corneal Keratinocytes. A clinical, histopathological, and ultrastructural study of a previously unreported family with MECD is presented. In this family the disease is ascribed to a novel mutation in KRT12. A molecular mechanism is proposed for MECD based on the comparison with other well characterised Keratin diseases.
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A novel Keratin 12 mutation in a German kindred with Meesmann's corneal dystrophy
The British journal of ophthalmology, 2000Co-Authors: Laura D. Corden, Ole Swensson, B. Swensson, R Rochels, B Wannke, Hans-jürgen Thiel, W.h.i. McleanAbstract:To study a kindred with Meesmann's corneal dystrophy (MCD) to determine if a mutation within the cornea specific K3 or K12 genes is responsible for the disease phenotype. Slit lamp examination of the cornea in four members of the kindred was carried out to confirm the diagnosis of MCD. The region encoding the helix initiation motif (HIM) of the K12 polypeptide was polymerase chain reaction (PCR) amplified from genomic DNA derived from affected individuals in the kindred. PCR products generated were subjected to direct automated sequencing. Restriction enzyme analysis employing Ban I was used to confirm the presence of the mutation in affected individuals of the family. Sequencing of the K12 gene in an affected individual from the family revealed a novel heterozygous missense mutation (413A-->C), predicting the substitution of a proline for a glutamine at codon 130 (Q130P) in the HIM of the K12 protein. The mutation was excluded from 50 normal, unaffected individuals by restriction enyzme analysis and was therefore unlikely to be a common polymorphism. A novel missense mutation in the K12 gene leads to MCD in a German kindred. Missense mutations have now been identified within the region encoding the helix initiation motif of the K12 protein in eight of 11 MCD kindreds analysed at the molecular level.
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a novel Keratin 12 mutation in a german kindred with meesmann s corneal dystrophy
British Journal of Ophthalmology, 2000Co-Authors: Laura D. Corden, Ole Swensson, B. Swensson, R Rochels, B Wannke, Hans-jürgen Thiel, W.h.i. McleanAbstract:AIM—To study a kindred with Meesmann's corneal dystrophy (MCD) to determine if a mutation within the cornea specific K3 or K12 genes is responsible for the disease phenotype. METHODS—Slit lamp examination of the cornea in four members of the kindred was carried out to confirm the diagnosis of MCD. The region encoding the helix initiation motif (HIM) of the K12 polypeptide was polymerase chain reaction (PCR) amplified from genomic DNA derived from affected individuals in the kindred. PCR products generated were subjected to direct automated sequencing. Restriction enzyme analysis employing Ban I was used to confirm the presence of the mutation in affected individuals of the family. RESULTS—Sequencing of the K12 gene in an affected individual from the family revealed a novel heterozygous missense mutation (413A→C), predicting the substitution of a proline for a glutamine at codon 130 (Q130P) in the HIM of the K12 protein. The mutation was excluded from 50 normal, unaffected individuals by restriction enyzme analysis and was therefore unlikely to be a common polymorphism. CONCLUSION—A novel missense mutation in the K12 gene leads to MCD in a German kindred. Missense mutations have now been identified within the region encoding the helix initiation motif of the K12 protein in eight of 11 MCD kindreds analysed at the molecular level.
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Molecular genetics of Meesmann's corneal dystrophy: ancestral and novel mutations in Keratin 12 (K12) and complete sequence of the human KRT12 gene.
Experimental eye research, 2000Co-Authors: Laura D. Corden, Ole Swensson, B. Swensson, R Rochels, Fjd Smith, Jouni Uitto, W.h.i. McleanAbstract:Recently, we identified the first mutations in corneal Keratins K3 and K12 in families with Meesmann's corneal dystrophy (MCD). Here, we sequenced all regions of the human K12 gene, to enable mutation detection for all exons using genomic DNA as a template. The human K12 genomic sequence spans 5919 bp and consists of eight exons. A microsatellite dinucleotide repeat was identified within intron 3, which was highly polymorphic and which we developed for use in genotype analysis. In addition, two mutations in the helix initiation motif of K12 were found in families with MCD. A novel mutation was detected in an American kindred, 410T-->C, which predicts the amino acid substitution M129T. In a German family, mutation 428G-->C was identified, predicting amino acid change R135T. The latter mutation was identical to that which we identified in the original kindred described by Meesmann. Using the intragenic microsatellite polymorphism in K12 and additional flanking markers, we were able to show that this family shares a common haplotype with the original Meesmann kindred. These results strongly imply that R135T represents an ancestral mutation in the German population. Both mutations occur in the highly conserved helix initiation motif of the K12 polypeptide. A total of eight mutations have now been reported in the K12 gene.
