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Genetic heterogeneity of pseudoxanthoma elasticum: the Chinese signature profile of ABCC6 and ENPP1 mutations.

Jin L, Jiang Q, Wu Z, Shao C, Zhou Y, Yang L, Uitto J, Wang G - J. Invest. Dermatol. (2015)

Bottom Line: Minimal rescue of the morpholino-induced phenotype was achieved with eight of the nine mutant human ABCC6 mRNAs tested, implying pathogenicity.This study demonstrates that the Chinese PXE population harbors unique ABCC6 mutations.These genetic data have implications for allele-specific therapy currently being developed for PXE.

View Article: PubMed Central - PubMed

Affiliation: Department of Dermatology, Xijing Hospital, Fourth Military Medical University, Xi'an, China.

ABSTRACT
Pseudoxanthoma elasticum (PXE), an autosomal recessive disorder characterized by ectopic mineralization, is caused by mutations in the ABCC6 gene. We examined clinically 29 Chinese PXE patients from unrelated families, so far the largest cohort of Asian PXE patients. In a subset of 22 patients, we sequenced ABCC6 and another candidate gene, ENPP1, and conducted pathogenicity analyses for each variant. We identified a total of 17 distinct mutations in ABCC6, 15 of them being, to our knowledge, previously unreported, including 5 frameshift and 10 missense variants. In addition, a missense mutation in combination with a recurrent nonsense mutation in ENPP1 was discovered in a pediatric PXE case. No cases with p.R1141X or del23-29 mutations, common in Caucasian patient populations, were identified. The 10 missense mutations in ABCC6 were expressed in the mouse liver via hydrodynamic tail-vein injections. One mutant protein showed cytoplasmic accumulation indicating abnormal subcellular trafficking, while the other nine mutants showed correct plasma membrane location. These nine mutations were further investigated for their pathogenicity using a recently developed zebrafish mRNA rescue assay. Minimal rescue of the morpholino-induced phenotype was achieved with eight of the nine mutant human ABCC6 mRNAs tested, implying pathogenicity. This study demonstrates that the Chinese PXE population harbors unique ABCC6 mutations. These genetic data have implications for allele-specific therapy currently being developed for PXE.

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Morphology of zebrafish 4 days after co-injection of an ABCC6A knock-down morpholino together with different human ABCC6 mRNA variantsThe morpholino-induced phenotype consisting of pericardiac edema, stunted growth and curled tail, similar to zebrafish injected with morpholino (MO) alone, was observed in zebrafish co-injected with human ABCC6 mRNA carrying p.R1141X, p.P4H, p.A9E, p.P21S, p.R419Q, p.E125K, p.E709G or p.L948P mutation, indicating lack of rescue and implying pathogenicity. Zebrafish co-injected with MO and ABCC6 mRNA carrying R64Q mutation showed wild-type phenotype, similar to fish injected with MO together with human wild-type (WT) ABCC6 mRNA.
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Figure 3: Morphology of zebrafish 4 days after co-injection of an ABCC6A knock-down morpholino together with different human ABCC6 mRNA variantsThe morpholino-induced phenotype consisting of pericardiac edema, stunted growth and curled tail, similar to zebrafish injected with morpholino (MO) alone, was observed in zebrafish co-injected with human ABCC6 mRNA carrying p.R1141X, p.P4H, p.A9E, p.P21S, p.R419Q, p.E125K, p.E709G or p.L948P mutation, indicating lack of rescue and implying pathogenicity. Zebrafish co-injected with MO and ABCC6 mRNA carrying R64Q mutation showed wild-type phenotype, similar to fish injected with MO together with human wild-type (WT) ABCC6 mRNA.

Mentions: The pathogenicity of the missense mutations identified in ABCC6 was further investigated in a zebrafish mRNA rescue assay that we have recently developed (Li et al., 2010a; Zhou et al., 2013). In this assay, zebrafish embryos are injected with an abcc6a morpholino which causes knock-down of the corresponding gene expression. As a consequence, the zebrafish embryos develop a profound phenotype consisting of pericardiac edema, stunted growth and curled tail, and the developing embryos die before the age of 7 days post fertilization (dpf) (Fig. 3). This phenotype can be fully rescued by injection of wild-type human ABCC6 mRNA together with the morpholino (Fig. 3). We consequently injected zebrafish embryos with the morpholino together with human ABCC6 mRNA harboring missense mutations identified in this study. As a negative control, the morpholino was injected with the human mRNA harboring stop codon mutation p.R1141X. As shown in Fig. 3, this mRNA containing the nonsense mutation did not rescue the phenotype. Injection of mutant mRNAs harboring the missense mutations identified in this study together with the morpholino revealed that 8 out of 9 mutations tested did not provide significant rescue as judged by either morphology of the zebrafish embryos (Fig. 3) or by the percent of lethality (Table 2) at 4 dpf, suggesting that they are pathogenic. Only one mutant mRNA, p.R64Q, resulted in rescue comparable to that of the wild-type mRNA (Fig. 3 and Table 2). However, the corresponding mutation, c.191G→A, was not present in the SNP database, and it is unclear whether this is a pathogenic mutation in the 5 patients with PXE.


Genetic heterogeneity of pseudoxanthoma elasticum: the Chinese signature profile of ABCC6 and ENPP1 mutations.

Jin L, Jiang Q, Wu Z, Shao C, Zhou Y, Yang L, Uitto J, Wang G - J. Invest. Dermatol. (2015)

Morphology of zebrafish 4 days after co-injection of an ABCC6A knock-down morpholino together with different human ABCC6 mRNA variantsThe morpholino-induced phenotype consisting of pericardiac edema, stunted growth and curled tail, similar to zebrafish injected with morpholino (MO) alone, was observed in zebrafish co-injected with human ABCC6 mRNA carrying p.R1141X, p.P4H, p.A9E, p.P21S, p.R419Q, p.E125K, p.E709G or p.L948P mutation, indicating lack of rescue and implying pathogenicity. Zebrafish co-injected with MO and ABCC6 mRNA carrying R64Q mutation showed wild-type phenotype, similar to fish injected with MO together with human wild-type (WT) ABCC6 mRNA.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4402129&req=5

Figure 3: Morphology of zebrafish 4 days after co-injection of an ABCC6A knock-down morpholino together with different human ABCC6 mRNA variantsThe morpholino-induced phenotype consisting of pericardiac edema, stunted growth and curled tail, similar to zebrafish injected with morpholino (MO) alone, was observed in zebrafish co-injected with human ABCC6 mRNA carrying p.R1141X, p.P4H, p.A9E, p.P21S, p.R419Q, p.E125K, p.E709G or p.L948P mutation, indicating lack of rescue and implying pathogenicity. Zebrafish co-injected with MO and ABCC6 mRNA carrying R64Q mutation showed wild-type phenotype, similar to fish injected with MO together with human wild-type (WT) ABCC6 mRNA.
Mentions: The pathogenicity of the missense mutations identified in ABCC6 was further investigated in a zebrafish mRNA rescue assay that we have recently developed (Li et al., 2010a; Zhou et al., 2013). In this assay, zebrafish embryos are injected with an abcc6a morpholino which causes knock-down of the corresponding gene expression. As a consequence, the zebrafish embryos develop a profound phenotype consisting of pericardiac edema, stunted growth and curled tail, and the developing embryos die before the age of 7 days post fertilization (dpf) (Fig. 3). This phenotype can be fully rescued by injection of wild-type human ABCC6 mRNA together with the morpholino (Fig. 3). We consequently injected zebrafish embryos with the morpholino together with human ABCC6 mRNA harboring missense mutations identified in this study. As a negative control, the morpholino was injected with the human mRNA harboring stop codon mutation p.R1141X. As shown in Fig. 3, this mRNA containing the nonsense mutation did not rescue the phenotype. Injection of mutant mRNAs harboring the missense mutations identified in this study together with the morpholino revealed that 8 out of 9 mutations tested did not provide significant rescue as judged by either morphology of the zebrafish embryos (Fig. 3) or by the percent of lethality (Table 2) at 4 dpf, suggesting that they are pathogenic. Only one mutant mRNA, p.R64Q, resulted in rescue comparable to that of the wild-type mRNA (Fig. 3 and Table 2). However, the corresponding mutation, c.191G→A, was not present in the SNP database, and it is unclear whether this is a pathogenic mutation in the 5 patients with PXE.

Bottom Line: Minimal rescue of the morpholino-induced phenotype was achieved with eight of the nine mutant human ABCC6 mRNAs tested, implying pathogenicity.This study demonstrates that the Chinese PXE population harbors unique ABCC6 mutations.These genetic data have implications for allele-specific therapy currently being developed for PXE.

View Article: PubMed Central - PubMed

Affiliation: Department of Dermatology, Xijing Hospital, Fourth Military Medical University, Xi'an, China.

ABSTRACT
Pseudoxanthoma elasticum (PXE), an autosomal recessive disorder characterized by ectopic mineralization, is caused by mutations in the ABCC6 gene. We examined clinically 29 Chinese PXE patients from unrelated families, so far the largest cohort of Asian PXE patients. In a subset of 22 patients, we sequenced ABCC6 and another candidate gene, ENPP1, and conducted pathogenicity analyses for each variant. We identified a total of 17 distinct mutations in ABCC6, 15 of them being, to our knowledge, previously unreported, including 5 frameshift and 10 missense variants. In addition, a missense mutation in combination with a recurrent nonsense mutation in ENPP1 was discovered in a pediatric PXE case. No cases with p.R1141X or del23-29 mutations, common in Caucasian patient populations, were identified. The 10 missense mutations in ABCC6 were expressed in the mouse liver via hydrodynamic tail-vein injections. One mutant protein showed cytoplasmic accumulation indicating abnormal subcellular trafficking, while the other nine mutants showed correct plasma membrane location. These nine mutations were further investigated for their pathogenicity using a recently developed zebrafish mRNA rescue assay. Minimal rescue of the morpholino-induced phenotype was achieved with eight of the nine mutant human ABCC6 mRNAs tested, implying pathogenicity. This study demonstrates that the Chinese PXE population harbors unique ABCC6 mutations. These genetic data have implications for allele-specific therapy currently being developed for PXE.

Show MeSH
Related in: MedlinePlus