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Cryptogenic stroke and small fiber neuropathy of unknown etiology in patients with alpha-galactosidase A -10T genotype.

Schelleckes M, Lenders M, Guske K, Schmitz B, Tanislav C, Ständer S, Metze D, Katona I, Weis J, Brand SM, Duning T, Brand E - Orphanet J Rare Dis (2014)

Bottom Line: Patients' mean GLA mRNA expression level was reduced to ~70% (p < 0.0001) and a dose-dependent effect of the -10T allele on GLA mRNA expression was observed in hemi/homozygous compared to heterozygous patients (p < 0.0001).Molecular analyzes revealed that the -10T allele resulted in a reduced promoter activity and an altered transcription factor binding, while a functional relevance of the co-segregated intronic variants was excluded by exon trapping.Based on this complementary approach of clinical observation and functional testing, we conclude that the GLA -10T allele could be causal for the observed neurological manifestations.

View Article: PubMed Central - PubMed

Affiliation: Internal Medicine D, Department of Nephrology, Hypertension and Rheumatology, University Hospital Muenster, Albert-Schweitzer-Campus 1, 48149, Muenster, Germany. Michael.Schelleckes@ukmuenster.de.

ABSTRACT

Background: Fabry disease (FD) is a multisystemic disorder with typical neurological manifestations such as stroke and small fiber neuropathy (SFN), caused by mutations of the alpha-galactosidase A (GLA) gene. We analyzed 15 patients carrying the GLA haplotype -10C>T [rs2071225], IVS2-81_-77delCAGCC [rs5903184], IVS4-16A>G [rs2071397], and IVS6-22C>T [rs2071228] for potential neurological manifestations.

Methods and results: Patients were retrospectively analyzed for stroke, transient ischemic attack (TIA), white matter lesions (WML) and SFN with neuropathic pain. Functional impact of the haplotype was determined by molecular genetic methods including real-time PCR, exon trapping, promoter deletion constructs and electrophoretic mobility shift assays. Symptomatic -10T allele carriers suffered from stroke, TIA, WML, and SFN with neuropathic pain. Patients' mean GLA mRNA expression level was reduced to ~70% (p < 0.0001) and a dose-dependent effect of the -10T allele on GLA mRNA expression was observed in hemi/homozygous compared to heterozygous patients (p < 0.0001). Molecular analyzes revealed that the -10T allele resulted in a reduced promoter activity and an altered transcription factor binding, while a functional relevance of the co-segregated intronic variants was excluded by exon trapping.

Conclusions: Based on this complementary approach of clinical observation and functional testing, we conclude that the GLA -10T allele could be causal for the observed neurological manifestations. Future studies are needed to clarify whether affected patients benefit from GLA enzyme replacement therapy for end-organ damage prevention.

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GLApromoter constructs are selectively activated by TFEB. (A) Representation of the four putative TFEB binding sites (underlined) in the GLA promoter. (B). Overexpression of TFEB in EA.hy926 cells (black bar) compared to mock transfected cells (white bar) and mutagenesis of conserved TFEB binding sites. (C) ChIP analysis in IHKE cells demonstrated the binding of TFEB. Input: Extracted chromatin served as positive control for PCR. Data are given as mean ± SEM. LU: light units; Luc: luciferase; ***p < 0.001.
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Fig7: GLApromoter constructs are selectively activated by TFEB. (A) Representation of the four putative TFEB binding sites (underlined) in the GLA promoter. (B). Overexpression of TFEB in EA.hy926 cells (black bar) compared to mock transfected cells (white bar) and mutagenesis of conserved TFEB binding sites. (C) ChIP analysis in IHKE cells demonstrated the binding of TFEB. Input: Extracted chromatin served as positive control for PCR. Data are given as mean ± SEM. LU: light units; Luc: luciferase; ***p < 0.001.

Mentions: To determine general regulatory factors involved in GLA expression regulation, we performed in silico analysis. We identified four putative TFEB binding sites within positions -333 to -274 from the translational start site (Figure 7A). Interestingly, Sardiello et al. [28] described TFEB as a “master regulator” of lysosomal genes. Subsequent overexpression of TFEB and GLA serial promoter deletion constructs in EA.hy926 cells resulted in a significant up to 4.5-fold increased (p < 0.001) transcriptional activity compared to the vector shuttle control (Figure 7B). Consequently, TFEB binding site mutation of two highly conserved binding sites led to total impairment of TFEB promoter activation (Figure 7B). Since this indicated a direct interaction of TFEB with the GLA promoter, we conducted ChIP experiments, which confirmed a specific interaction of TFEB with region -425 to -239 of the GLA promoter (Figure 7C).Figure 7


Cryptogenic stroke and small fiber neuropathy of unknown etiology in patients with alpha-galactosidase A -10T genotype.

Schelleckes M, Lenders M, Guske K, Schmitz B, Tanislav C, Ständer S, Metze D, Katona I, Weis J, Brand SM, Duning T, Brand E - Orphanet J Rare Dis (2014)

GLApromoter constructs are selectively activated by TFEB. (A) Representation of the four putative TFEB binding sites (underlined) in the GLA promoter. (B). Overexpression of TFEB in EA.hy926 cells (black bar) compared to mock transfected cells (white bar) and mutagenesis of conserved TFEB binding sites. (C) ChIP analysis in IHKE cells demonstrated the binding of TFEB. Input: Extracted chromatin served as positive control for PCR. Data are given as mean ± SEM. LU: light units; Luc: luciferase; ***p < 0.001.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4255940&req=5

Fig7: GLApromoter constructs are selectively activated by TFEB. (A) Representation of the four putative TFEB binding sites (underlined) in the GLA promoter. (B). Overexpression of TFEB in EA.hy926 cells (black bar) compared to mock transfected cells (white bar) and mutagenesis of conserved TFEB binding sites. (C) ChIP analysis in IHKE cells demonstrated the binding of TFEB. Input: Extracted chromatin served as positive control for PCR. Data are given as mean ± SEM. LU: light units; Luc: luciferase; ***p < 0.001.
Mentions: To determine general regulatory factors involved in GLA expression regulation, we performed in silico analysis. We identified four putative TFEB binding sites within positions -333 to -274 from the translational start site (Figure 7A). Interestingly, Sardiello et al. [28] described TFEB as a “master regulator” of lysosomal genes. Subsequent overexpression of TFEB and GLA serial promoter deletion constructs in EA.hy926 cells resulted in a significant up to 4.5-fold increased (p < 0.001) transcriptional activity compared to the vector shuttle control (Figure 7B). Consequently, TFEB binding site mutation of two highly conserved binding sites led to total impairment of TFEB promoter activation (Figure 7B). Since this indicated a direct interaction of TFEB with the GLA promoter, we conducted ChIP experiments, which confirmed a specific interaction of TFEB with region -425 to -239 of the GLA promoter (Figure 7C).Figure 7

Bottom Line: Patients' mean GLA mRNA expression level was reduced to ~70% (p < 0.0001) and a dose-dependent effect of the -10T allele on GLA mRNA expression was observed in hemi/homozygous compared to heterozygous patients (p < 0.0001).Molecular analyzes revealed that the -10T allele resulted in a reduced promoter activity and an altered transcription factor binding, while a functional relevance of the co-segregated intronic variants was excluded by exon trapping.Based on this complementary approach of clinical observation and functional testing, we conclude that the GLA -10T allele could be causal for the observed neurological manifestations.

View Article: PubMed Central - PubMed

Affiliation: Internal Medicine D, Department of Nephrology, Hypertension and Rheumatology, University Hospital Muenster, Albert-Schweitzer-Campus 1, 48149, Muenster, Germany. Michael.Schelleckes@ukmuenster.de.

ABSTRACT

Background: Fabry disease (FD) is a multisystemic disorder with typical neurological manifestations such as stroke and small fiber neuropathy (SFN), caused by mutations of the alpha-galactosidase A (GLA) gene. We analyzed 15 patients carrying the GLA haplotype -10C>T [rs2071225], IVS2-81_-77delCAGCC [rs5903184], IVS4-16A>G [rs2071397], and IVS6-22C>T [rs2071228] for potential neurological manifestations.

Methods and results: Patients were retrospectively analyzed for stroke, transient ischemic attack (TIA), white matter lesions (WML) and SFN with neuropathic pain. Functional impact of the haplotype was determined by molecular genetic methods including real-time PCR, exon trapping, promoter deletion constructs and electrophoretic mobility shift assays. Symptomatic -10T allele carriers suffered from stroke, TIA, WML, and SFN with neuropathic pain. Patients' mean GLA mRNA expression level was reduced to ~70% (p < 0.0001) and a dose-dependent effect of the -10T allele on GLA mRNA expression was observed in hemi/homozygous compared to heterozygous patients (p < 0.0001). Molecular analyzes revealed that the -10T allele resulted in a reduced promoter activity and an altered transcription factor binding, while a functional relevance of the co-segregated intronic variants was excluded by exon trapping.

Conclusions: Based on this complementary approach of clinical observation and functional testing, we conclude that the GLA -10T allele could be causal for the observed neurological manifestations. Future studies are needed to clarify whether affected patients benefit from GLA enzyme replacement therapy for end-organ damage prevention.

Show MeSH
Related in: MedlinePlus