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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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Influence of intronic variants on GLA processing. (A) Localization of the intronic variants IVS2-81_-77delCAGCC [rs5903184]), IVS4-16A>G [rs2071397] and IVS6-22C>T [rs2071228]). (B)GLA exons and denoted flanking introns (wild-type and variant). (C) Agarose gel separation of exon trapping products. wt: wild-type.
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Fig6: Influence of intronic variants on GLA processing. (A) Localization of the intronic variants IVS2-81_-77delCAGCC [rs5903184]), IVS4-16A>G [rs2071397] and IVS6-22C>T [rs2071228]). (B)GLA exons and denoted flanking introns (wild-type and variant). (C) Agarose gel separation of exon trapping products. wt: wild-type.

Mentions: As intronic GLA variants such as IVS4+919G>A have been reported to cause late-onset FD with cardiac phenotype by altering GLA mRNA processing [7,23], we analyzed the potential effect of the intronic variants (IVS2-81_-77delCAGCC; IVS4-16A>G and IVS6-22C>T) co-segregating with the -10T allele by exon trapping experiments in an endothelial cell line (Figure 6). For all investigated intronic variants neither differences in transcript length nor sequence were detected. These observations suggest that the intronic variants IVS2-81_-77, IVS4-16 and IVS6-22 have no effect on GLA mRNA processing in contrast to the positive control IVS4+919G>A (Figure 6C). These results were confirmed in a neuronal cell line (data not shown). After exclusion of the functional impact of the intronic variants the reduction of GLA mRNA expression should be assigned to the -10T promoter allele.Figure 6


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)

Influence of intronic variants on GLA processing. (A) Localization of the intronic variants IVS2-81_-77delCAGCC [rs5903184]), IVS4-16A>G [rs2071397] and IVS6-22C>T [rs2071228]). (B)GLA exons and denoted flanking introns (wild-type and variant). (C) Agarose gel separation of exon trapping products. wt: wild-type.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig6: Influence of intronic variants on GLA processing. (A) Localization of the intronic variants IVS2-81_-77delCAGCC [rs5903184]), IVS4-16A>G [rs2071397] and IVS6-22C>T [rs2071228]). (B)GLA exons and denoted flanking introns (wild-type and variant). (C) Agarose gel separation of exon trapping products. wt: wild-type.
Mentions: As intronic GLA variants such as IVS4+919G>A have been reported to cause late-onset FD with cardiac phenotype by altering GLA mRNA processing [7,23], we analyzed the potential effect of the intronic variants (IVS2-81_-77delCAGCC; IVS4-16A>G and IVS6-22C>T) co-segregating with the -10T allele by exon trapping experiments in an endothelial cell line (Figure 6). For all investigated intronic variants neither differences in transcript length nor sequence were detected. These observations suggest that the intronic variants IVS2-81_-77, IVS4-16 and IVS6-22 have no effect on GLA mRNA processing in contrast to the positive control IVS4+919G>A (Figure 6C). These results were confirmed in a neuronal cell line (data not shown). After exclusion of the functional impact of the intronic variants the reduction of GLA mRNA expression should be assigned to the -10T promoter allele.Figure 6

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