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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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Related in: MedlinePlus

Functional analysis of the -10T allele. (A) Representative chromatograms showing nucleotide substitution at position -10. (B) Carriers of the minor -10T allele with neurological manifestations (black box; n = 11) showed significantly decreased GLA mRNA expression levels in peripheral mononuclear blood cells compared to healthy non-carriers (white box; male: n = 8; female: n = 9). (C) GLA expression is significantly decreased in symptomatic homo-/hemizygous T allele carriers (n = 4) versus symptomatic CT carriers (n = 7). (D) Schematic representation of the GLA 5′-flanking region. (E) Transient transfection of GLA promoter constructs in EA.hy926 cells revealed two regions with significant transcriptional activity. (F-H) Insertion of the minor T allele into promoter constructs (black bars) resulted in a decreased transcriptional activity in SH-SY5Y (F), EA.hy926 (G) and THP-1 (H) cells. (I) EMSA with nuclear extract from EA.hy926 cells revealed one specific competable (non-allelic) band (upper arrow) and a slower migrating T allele-specific band (lower arrow). F: probe without extract and competitor. Data are given as mean ± SEM; LU: light units; Luc: luciferase.*p < 0.05; ***p < 0.001.
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Fig5: Functional analysis of the -10T allele. (A) Representative chromatograms showing nucleotide substitution at position -10. (B) Carriers of the minor -10T allele with neurological manifestations (black box; n = 11) showed significantly decreased GLA mRNA expression levels in peripheral mononuclear blood cells compared to healthy non-carriers (white box; male: n = 8; female: n = 9). (C) GLA expression is significantly decreased in symptomatic homo-/hemizygous T allele carriers (n = 4) versus symptomatic CT carriers (n = 7). (D) Schematic representation of the GLA 5′-flanking region. (E) Transient transfection of GLA promoter constructs in EA.hy926 cells revealed two regions with significant transcriptional activity. (F-H) Insertion of the minor T allele into promoter constructs (black bars) resulted in a decreased transcriptional activity in SH-SY5Y (F), EA.hy926 (G) and THP-1 (H) cells. (I) EMSA with nuclear extract from EA.hy926 cells revealed one specific competable (non-allelic) band (upper arrow) and a slower migrating T allele-specific band (lower arrow). F: probe without extract and competitor. Data are given as mean ± SEM; LU: light units; Luc: luciferase.*p < 0.05; ***p < 0.001.

Mentions: In detail, two patients (#12 and #13) suffered from stroke and three patients (#7, #8, and #10) from TIA (Table 1). In addition to the index patient, skin biopsies of three additional patients (#2, #7, and #11) revealed SFN diagnosed by reduction of IENFD (#1: 4.64/mm, #2: 4.40/mm, #7: 4.65/mm, #11: 3.70/mm; Figure 2D,G), while the number of focal axonal swelling of intra-epidermal axons was increased by 20% in patient #2 (Figure 2E,F). To evaluate the somatosensory phenotype of pain, QST was performed and was abnormal in 8 patients (#1-4, #7, and #9-11; Table 1), indicating functional impairment of A-delta- and C-fibers. In patients #1, #2, #4 and #7 DMA was present, and patients #3, #7, #9 and #10 had PHS. The Z-score profile of -10T allele carriers showed a predominant loss of sensory function in terms of cold and warm hypoesthesia (CDT, WDT, and TSL) and a reduced mechanical and vibratory sensation (MDT and VDT), a profile consistent with a selective small fiber neuropathy in FD (Figure 3) [27]. Available transmission electron microscopy of skin biopsies from patients #6 and #7 showed irregular, only partially concentric lamellar lysosomal inclusions devoid of strictly parallel membranes (Figure 4). Since the -10T haplotype may result in decreased mRNA expression we performed real-time PCR analysis revealing decreased mRNA expression levels in minor T allele carriers. This haplotype resulted in a significant GLA mRNA expression decrease of 30% compared to healthy controls (n = 17; p < 0.0001; Figure 5B). The observed mRNA reduction was associated with reduced enzymatic GLA activity in three patients (#3, #11 and #12; Table 2).Figure 3


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)

Functional analysis of the -10T allele. (A) Representative chromatograms showing nucleotide substitution at position -10. (B) Carriers of the minor -10T allele with neurological manifestations (black box; n = 11) showed significantly decreased GLA mRNA expression levels in peripheral mononuclear blood cells compared to healthy non-carriers (white box; male: n = 8; female: n = 9). (C) GLA expression is significantly decreased in symptomatic homo-/hemizygous T allele carriers (n = 4) versus symptomatic CT carriers (n = 7). (D) Schematic representation of the GLA 5′-flanking region. (E) Transient transfection of GLA promoter constructs in EA.hy926 cells revealed two regions with significant transcriptional activity. (F-H) Insertion of the minor T allele into promoter constructs (black bars) resulted in a decreased transcriptional activity in SH-SY5Y (F), EA.hy926 (G) and THP-1 (H) cells. (I) EMSA with nuclear extract from EA.hy926 cells revealed one specific competable (non-allelic) band (upper arrow) and a slower migrating T allele-specific band (lower arrow). F: probe without extract and competitor. Data are given as mean ± SEM; LU: light units; Luc: luciferase.*p < 0.05; ***p < 0.001.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig5: Functional analysis of the -10T allele. (A) Representative chromatograms showing nucleotide substitution at position -10. (B) Carriers of the minor -10T allele with neurological manifestations (black box; n = 11) showed significantly decreased GLA mRNA expression levels in peripheral mononuclear blood cells compared to healthy non-carriers (white box; male: n = 8; female: n = 9). (C) GLA expression is significantly decreased in symptomatic homo-/hemizygous T allele carriers (n = 4) versus symptomatic CT carriers (n = 7). (D) Schematic representation of the GLA 5′-flanking region. (E) Transient transfection of GLA promoter constructs in EA.hy926 cells revealed two regions with significant transcriptional activity. (F-H) Insertion of the minor T allele into promoter constructs (black bars) resulted in a decreased transcriptional activity in SH-SY5Y (F), EA.hy926 (G) and THP-1 (H) cells. (I) EMSA with nuclear extract from EA.hy926 cells revealed one specific competable (non-allelic) band (upper arrow) and a slower migrating T allele-specific band (lower arrow). F: probe without extract and competitor. Data are given as mean ± SEM; LU: light units; Luc: luciferase.*p < 0.05; ***p < 0.001.
Mentions: In detail, two patients (#12 and #13) suffered from stroke and three patients (#7, #8, and #10) from TIA (Table 1). In addition to the index patient, skin biopsies of three additional patients (#2, #7, and #11) revealed SFN diagnosed by reduction of IENFD (#1: 4.64/mm, #2: 4.40/mm, #7: 4.65/mm, #11: 3.70/mm; Figure 2D,G), while the number of focal axonal swelling of intra-epidermal axons was increased by 20% in patient #2 (Figure 2E,F). To evaluate the somatosensory phenotype of pain, QST was performed and was abnormal in 8 patients (#1-4, #7, and #9-11; Table 1), indicating functional impairment of A-delta- and C-fibers. In patients #1, #2, #4 and #7 DMA was present, and patients #3, #7, #9 and #10 had PHS. The Z-score profile of -10T allele carriers showed a predominant loss of sensory function in terms of cold and warm hypoesthesia (CDT, WDT, and TSL) and a reduced mechanical and vibratory sensation (MDT and VDT), a profile consistent with a selective small fiber neuropathy in FD (Figure 3) [27]. Available transmission electron microscopy of skin biopsies from patients #6 and #7 showed irregular, only partially concentric lamellar lysosomal inclusions devoid of strictly parallel membranes (Figure 4). Since the -10T haplotype may result in decreased mRNA expression we performed real-time PCR analysis revealing decreased mRNA expression levels in minor T allele carriers. This haplotype resulted in a significant GLA mRNA expression decrease of 30% compared to healthy controls (n = 17; p < 0.0001; Figure 5B). The observed mRNA reduction was associated with reduced enzymatic GLA activity in three patients (#3, #11 and #12; Table 2).Figure 3

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