A Novel Homozygous Missense Mutation in <i><scp>HOXC</scp>13</i> Leads to Autosomal Recessive Pure Hair and Nail Ectodermal Dysplasia

Li, Xiaoxiao; Orseth, Meredith L.; Smith, J. Michael; Brehm, Mary Abigail; Agim, Nnenna G.; Glass, Donald A.

doi:10.1111/pde.13074

Cited by 7 publications

(10 citation statements)

References 15 publications

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“…28 A previous study has shown that the p.Q271R missense mutation in Hoxc13 gene causes the PHNED and the highly conserved coding sequence between humans and rabbits ( Figure 4A,B). 28 A previous study has shown that the p.Q271R missense mutation in Hoxc13 gene causes the PHNED and the highly conserved coding sequence between humans and rabbits ( Figure 4A,B).…”

Section: A-to-g Base Conversion At Hoxc13 To Mimic Human Phned Mutamentioning

confidence: 81%

“…In addition, the NG‐ABEmax exhibited average base editing frequencies from 9.30% ± 3.83% to 83.37% ± 5.78% with a similar ~6 nt editing window (Figures C‐H, S5 and Table ). These target loci with A·T to G·C conversion in Psen1 (p.M146V), Sod1 (p.I151T), Lmna‐2 (p.L530P), and Hoxc13 (p.Q271R) have been reported to cause Alzheimer's disease (AD), amyotrophic lateral sclerosis (ALS), Emery‐Dreifuss muscular dystrophy (EDMD), and Pure hair and nail ectodermal dysplasia (PHNED), respectively (Figures B, S6). These results indicate that NG‐ABEmax is efficient at relaxed NG PAMs in rabbit embryos, suggesting the great potential of this system to develop animal models for precisely mimic human genetic diseases.…”

Section: Resultsmentioning

confidence: 99%

“…Targeted A-to-G conversions were observed in all target loci, with high efficiency from 80% to 100% (Figures 3C-H, S5 and Table 2). These target loci with A·T to G·C conversion in Psen1 (p.M146V), 25 Sod1 (p.I151T), 26 Lmna-2 (p.L530P), 27 and Hoxc13 (p.Q271R) 28 have been reported to cause Alzheimer's disease (AD), amyotrophic lateral sclerosis (ALS), Emery-Dreifuss muscular dystrophy (EDMD), and Pure hair and nail ectodermal dysplasia (PHNED), respectively ( Figures 3B, S6). A, Schematic representation of the NG-BE4max system.…”

Section: Ng-abemax In Rabbit Embryosmentioning

confidence: 99%

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Highly efficient base editing with expanded targeting scope using SpCas9‐NG in rabbits

et al. 2019

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Base editors, composed of a cytidine deaminase or an evolved adenine deaminase fused to Cas9 nickase, enable efficient C-to-T or A-to-G conversion in various organisms. However, the NGG protospacer adjacent motif (PAM) requirement of Streptococcus pyogenes Cas9 (SpCas9) substantially limits the target sites suitable for base editing. Quite recently, a new engineered SpCas9-NG variant, which can recognize minimal NG PAMs more efficiently than the present xCas9 variant. Here, we investigated the efficiency and PAM compatibility of SpCas9-NG-assisted cytidine base editors (CBEs) and adenine base editors (ABEs) in rabbits. In this study, we showed that NG-BE4max and NG-ABEmax systems can achieve a targeted mutation efficiency of 75%-100% and 80%-100% with excellent PAM compatibility of NGN PAMs in rabbit embryos, respectively. In addition, both base editors were successfully applied to create new rabbit models with precise point mutations, demonstrating their high efficiency and expanded genome-targeting scope in rabbits.Meanwhile, NG-ABEmax can be used to precisely mimic human Hoxc13 p.Q271R missense mutation in Founder (F0) rabbits, which is arduous for conventional ABEs to achieve due to a NGA PAM requirement. Collectively, NG-BE4max and NG-ABEmax systems provide promising tools to perform efficient base editing with expanded targeting scope in rabbits and enhances its capacity to model human diseases. K E Y W O R D Sbase editor, CRISPR/Cas9, NG PAMs, rabbit | 589 LIU et aL.

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Section: A-to-g Base Conversion At Hoxc13 To Mimic Human Phned Mutamentioning

confidence: 81%

Section: Resultsmentioning

confidence: 99%

Section: Ng-abemax In Rabbit Embryosmentioning

confidence: 99%

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Highly efficient base editing with expanded targeting scope using SpCas9‐NG in rabbits

et al. 2019

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“…Most of these mutations (Tyr130*, Leu119Trpfs 20, His68Glnfs*84, Ser135*, 27.6 kb deletion) are nonsense mutations. Interestingly, two missense mutations in the homeodomain of HOXC13 caused ectodermal dysplasia [ 36 , 37 ]. However, the mechanism was unclear.…”

Section: Discussionmentioning

confidence: 99%

“…To date, seven mutations had been reported in this gene causing pure hair and nail ectodermal dysplasia (PHNED), which led to complete hair loss and nail dysplasia (Table 1 ) [ 33 – 35 ]. Interestingly, two single nucleotide polymorphisms (SNPs) (c.812A > G and c.929A > C) in the homeodomain that belong to missense mutation caused ectodermal dysplasia [ 36 , 37 ]. However, the mechanism was still unknown.…”

Section: Introductionmentioning

confidence: 99%

The inconsistent regulation of HOXC13 on different keratins and the regulation mechanism on HOXC13 in cashmere goat (Capra hircus)

et al. 2018

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BackgroundDuring hair growth, cortical cells emerging from the proliferative follicle bulb rapidly undergo a differentiation program and synthesize large amounts of hair keratin proteins. In this process, HOXC13 is one critical regulatory factor, proved by the hair defects in HOXC13 mutant mice and HOXC13 mutant patients. However, inconsistent conclusions were drawn from previous researches regarding the regulation of HOXC13 on different keratins. Whether HOXC13 has extensive and unified regulatory role on these numerous keratins is unclear.ResultsIn this study, firstly, RNA-seq was performed to reveal the molecular mechanism of cashmere cycle including anagen and telogen. Subsequently, combining the sequencing with qRT-PCR and immunofluorescent staining results, we found that HOXC13 showed similar expression pattern with a large proportion of keratins except for KRT1 and KRT2, which were higher in anagen compared with telogen. Then, the regulatory role of HOXC13 on different keratins was investigated using dual-luciferase reporter system and keratin promoter-GFP system by overexpressing HOXC13 in HEK 293 T cells and dermal papilla cells. Our results demonstrated that HOXC13 up-regulated the promoter activity of KRT84 and KRT38, while down-regulated the promoter activity of KRT1 and KRT2, which suggested HOXC13 had an ambivalent effect on the promoters of different KRTs. Furtherly, the regulation on HOXC13 itself was investigated. At transcriptional level, the binding sites of HOXC13 and LEF1 were found in the promoter of HOXC13. Then, through transfecting corresponding overexpression vector and dual-luciferase reporter system into dermal papilla cells, the negative-feedback regulation of HOXC13 itself and positive regulation of LEF1 on HOXC13 promoter were revealed. In addition, melatonin could significantly increase the promoter activity of HOXC13 under the concentration of 10 μM and 25 μM by adding exogenous melatonin into dermal papilla cells. At post-transcriptional level, we investigated whether chi-miR-200a could target HOXC13 through dual-luciferase reporter system. At epigenetic level, we investigated the methylation level of HOXC13 promoter at different stages including anagen, telogen and 60d of embryonic period. As a result, miR-200a and methylation were not regulatory factors of HOXC13. Interestingly, we found two SNPs (c.812A > G and c.929A > C) in the homeodomain of HOXC13 that could deprive the regulatory function of HOXC13 on keratins without changing its protein expression.ConclusionHOXC13 had an inconsistent effect on the promoters of different keratins. Two SNPs (c.812A > G and c.929A > C) in the homeodomain of HOXC13 deprived its function on keratin regulation. Besides, the negative-feedback regulation by HOXC13 itself and positive regulation by LEF1 and melatonin on HOXC13 promoter were revealed. This study will enrich the function of HOXC13 on keratin regulation and contribute to understand the mechanism of hair follicle differentiation.Electronic supplementary materialThe online version of...

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Gene cloning and seamless site-directed mutagenesis using single-strand annealing (SSA)

Luo

Wang

Jiao

et al. 2018

Appl Microbiol Biotechnol

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The full length of interested genes can be usually cloned by assembling exons or RACE products through overlap PCR. However, the procedure requires multiple PCR steps, which are prone to random mutagenesis. Here, we present a novel SSA-based method for gene cloning and seamless site-directed mutagenesis. We firstly cloned the full-length coding sequence of Cashmere goat (Capra hircus) Hoxc13 gene by assembling exons amplified from genomic DNA. Secondly, we created a Hoxc13 loss-function mutant seamlessly and further illustrated that direct repeat length of 25 bp is enough to trigger the SSA repair in routine E. coli strains including DH5α, Trans1t1, JM109, and Top10. Moreover, we cloned another full-length mutant of Foxn1 gene from Cashmere goat cDNA using further shortened direct repeats of 19 bp. In summary, our study provided an alternative method to overcome the difficulties during overlap PCR in some particular cases for gene cloning.

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A Novel Homozygous Missense Mutation in HOXC13 Leads to Autosomal Recessive Pure Hair and Nail Ectodermal Dysplasia

Cited by 7 publications

References 15 publications

Highly efficient base editing with expanded targeting scope using SpCas9‐NG in rabbits

Highly efficient base editing with expanded targeting scope using SpCas9‐NG in rabbits

The inconsistent regulation of HOXC13 on different keratins and the regulation mechanism on HOXC13 in cashmere goat (Capra hircus)

Gene cloning and seamless site-directed mutagenesis using single-strand annealing (SSA)

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