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

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.

Figure 1

Presentation of the retrospective study design. Between 07/2011 and 12/2013 86 new patients presented at the Fabry center of the University Hospital Muenster with Fabry-typical neurological manifestations such as small fiber neuropathy with neuropathic pain, or stroke/TIA of unknown etiology. Out of 86 patients 49 had classical Fabry disease-causing mutations, 12 patients were symptomatic -10T allele carriers and 25 patients had no GLA mutation. Two asymptomatic patients were identified by family screening. We analyzed GLA expression in all detected -10T allele carriers and retrospectively analyzed clinical data.

Figure 2

Magnetic resonance imaging (MRI) and small fiber neuropathy diagnosis by PGP9.5 immunofluorescence. (A-C) Fluid attenuated inversion recovery (FLAIR) MRI of patient #1 showed multiple, punctuated white matter lesions from periventricular (yellow arrows and circles) to subcortical (red arrows and circles) without gadolinium enhancement. (D) Skin biopsy analysis of patient #2 showed the reduction of the small epidermal nerve fibers (arrow). Scale bar = 20 μm. (E-F). The number of focal axonal swellings (arrows) larger than 1.5 μm was increased. Scale bar = 10 μm. (G) Skin biopsy of a healthy control. Scale bar = 30 μm.

Figure 3

Z-score sensory profiles of -10T allele carriers. The Z-score profile of -10T allele carriers shows 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). The profile is consistent with a selective small fiber neuropathy in FD. Negative Z-scores indicate loss of sensation, positive Z-scores indicate gain of sensation. Error bars represent standard deviation of the mean; n = 10. CDT: cold detection threshold, WDT: warm detection threshold, TSL: thermal sensory limen, CPT: cold pain threshold, HPT: heat pain threshold, PPT: pressure pain threshold, MPT: mechanical pain threshold, MPS: mechanical pain sensitivity, WUR: wind-up ratio, MDT: mechanical detection threshold, VDT: vibration detection threshold.

Figure 4

Transmission electron microscopy (TEM) of skin biopsy of patient #6 (A) and #7 (B). TEM of skin biopsies showed some endothelia of capillaries with irregular lamellar inclusions (ML). Mitochondria (M). Nucleus (N). Lumen of the capillary (L). Scale bar = 500 nm.

Figure 5

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.

Figure 6

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.

Figure 7

GLA promoter 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.