Only a few cases of intronic haplotypes accompanied by classical symptoms of Fabry disease have been reported [6,7,8, 10, 12,13,14,15]. Fabry disease phenotype-related intronic haplotype variants are rare, and it is unclear whether intronic variants of the GLA gene correlate with Fabry disease. However, there are several ways that non-coding intronic variants could induce the clinical symptoms of Fabry disease. First, recent studies have demonstrated decreases in GLA enzyme expression in patients with the c.−10 C > T (rs2071225) variant located in the GLA promoter [6, 12, 15]. Similar to our case of CIH variants, mutation of the promoter region can decrease GLA expression, resulting in classic manifestations of Fabry disease [12, 13]. Second, intronic variations can affect alternative pre-mRNA splicing. Ishii et al. (10) showed that abnormal splicing in the mid-intronic GLA mutation of IVS4 + 919 A > G increased the alternatively spliced α-galactosidase A transcript, resulting in the Fabry cardiac phenotype. In fact, changes in GLA gene splicing were observed in the two intronic variants of c.370–81_370–77delCAGCC (rs5903184) and c.640–16 A > G (rs2071397) among the patients with CIH mutations [6]. Fabry disease has extensive phenotypic heterogeneity, even within patients with the same variants. The phenotype is probably modified not only by genetic factors but also epigenetics or factors unrelated to GLA [16]. The c.−10 C > T (rs2071397) variant is situated in a CpG island that contributes to epigenetic mechanisms by being the site of DNA methylation [17]. DNA methylation is a universal epigenetic mechanism for modifying gene expression. Thus, epigenetic changes may aberrantly reduce GLA gene expression when combined with this prevalent CIH sequence [15]. However, an additive effect of a number of intronic variants has been suggested by a previous report, although it was not conclusive [15].
Intron variants such as CIH variants may not be diagnosed using traditional exon-oriented sequencing. Our patient also had symptoms and clinical manifestations of Fabry disease but had difficulties in diagnosis. All the other GLA polymorphisms that constitute this CIH are relatively close to exon/intron boundaries, with the exception of c.369 + 990 C > A (rs1023431). Their detection in routine Sanger sequencing for the genetic diagnosis of Fabry disease depends on the position of the polymerase chain reaction (PCR) primers used. This variability in primer placement is one of the reasons why this CIH has been inconsistently described in the literature. In patients suspected of having a genetic disease due to a typical course or family history, but for whom an exonic mutation is not found, it may be necessary to perform intronic gene analysis. In recent studies, there have been reports of detecting novel pathogenic intronic variants using long-read sequencing [18]. If such techniques are introduced to diagnosis of Fabry disease, it will explain an important part of the missing heritability even in patients whose causative variants has not been diagnosed using methods such as Sanger sequencing or new generation sequencing. The different clinical course between the twin sisters in our case seems to be understandable in this context. Even if it is not limited to twins, intrafamilial variability is common in Fabry disease, and this is an important cause of delay in diagnosis and treatment of Fabry disease [19]. Therefore, following the diagnosis of Fabry disease, complete family screening is necessary, and it is important to observe the development of clinical symptoms through long-term follow-up.
There have been several reported cases of Fabry disease in female monozygotic twins. Discordant phenotypes in females with Fabry disease who have the same variants are explained by an imbalance in maternal and paternal X-chromosome expression. The separation of the fertilized egg into two monozygotes occurs before the second week of embryonic life, with X-chromosome inactivation happening around day 16, in a random basis [3]. Therefore, variable phenotypic expression can be observed in monozygotic female twins with X-linked disorder like Fabry disease [2,3,4]. In previously reported cases of female monozygotic twins, the twin sister of the proband typically did not develop clinical symptom of Fabry disease. However, in our case, the monozygotic twin sister of the proband exhibited similar clinical phenotypes, including cardiac, cerebrovascular, and kidney manifestations of Fabry disease. Furthermore, this is the first reported case caused by CIH variants.
ERT has long been the main therapeutic strategy for patients with Fabry disease. There have been several case reports of patients with CIH variants treated with ERT [7, 9, 12, 20]. A symptomatic German female − 10T allele carrier started treatment with agalsidase-β, and this led to clinical stabilization of Fabry disease symptoms and a significant reduction in neuropathic pain [9]. Interestingly, reducing the enzyme dose to half resulted in increased pain and reduced physical activity. In another study, two female Spanish patients with 5 CIH variants who presented with cerebrovascular disease and/or acroparesthesias achieved clinical stability after ERT [20]. Our index patient received ERT, while her twin sister did not. Two years of ERT may have stabilized the disease and reduced the abdominal pain in the patient. Therefore, it is necessary to observe how ERT or not having ERT will affect long-term prognoses in these monozygotic twins with the same genetic background. However, in terms of reduction in GFR, a decline in renal function was observed despite ERT, which is presumed to be due to the delayed diagnosis of Fabry disease in this patient and the delayed start of ERT. Wanner et al. reported that the lower baseline eGFR was associated with renal disease progression in women with Fabry disease [21]. Even in patients who underwent ERT, it was reported that the eGFR slope was steeper when baseline renal injury was severe [22]. Our patient had moderate-degree interstitial fibrosis and tubular atrophy observed in a renal biopsy 2 years before diagnosis, and it is presumed that her renal function deteriorated despite ERT. Lastly, the ERT period was relatively short in our patient, so the effect of ERT does not seem to differ dramatically between twins yet, but long-term follow-up is needed in the future.
Our study has limitations. First, we could not perform the functional study but there are some results of the previous studies which tested the functional effects of the CIH variants. Gervas-Arruga et al. reported that CIH carriers exhibited altered GLA expression, despite most carriers having high residual enzyme activity [6]. In addition, c.-10 C > T (rs2071225) variant which is located in the promotor region was shown to have decreased protein binding capacity in the EMSA study. Furthermore, Zeevi et al. reported that the patients with intronic variants demonstrated reduced mRNA expression of the GLA gene, suggesting a potential additive effect of these intronic variants [15]. We believe that these findings provide indirect evidence of the functional impact of CIH variants. Second, we were unable to confirm the nature of lamellated lipid inclusions in podocytes observed on EM. However, in previous studies, the accumulation of Gb3 was confirmed through methods such as anti-CD77 fluorescence in skin biopsy specimens of patients or in vitro studies with CIH variants [6, 8]. Third, despite the various evidence presented in this study and previous studies, it remains unclear whether CIH variants are truly pathogenic. Further research will likely be required, including the continuous accumulation of data on the relationship between intronic variants and phenotypes, as well as the application of new research techniques. Nevertheless, this is the first reported case of female monozygotic twins with CIH variants manifesting cardiac, cerebrovascular, and renal symptoms suggestive of Fabry disease. Our findings suggest that intronic gene analysis may be necessary in patients without exonic variants who exhibit Fabry disease symptoms.
Add Comment