A New Class of Pathogenic Non-Coding Variants in GLA—LRS Reveals Non-Coding Variants in Fabry Disease

1. Introduction

Fabry disease (FD; OMIM 301500) is a rare X-linked lysosomal storage disease caused by deleterious variants in GLA (NCBI, GRCh37.p13, NC_000023.10), which encodes the enzyme alpha-galactosidase A (α-Gal A; EC 3.2.1.22)[1]. This leads to accumulation of its substrate, globotriaosylceramide (Gb3), its deacylated metabolite globotriaosylsphingosine (lyso-Gb3), and related analogs[1,2]. Progressive accumulation of Gb3 and lyso-Gb3 within lysosomes across a variety of cell types and organs disrupts cellular dysfunction, leading to serious complications such as renal failure, heart failure, and stroke, and ultimately reduces life expectancy[3,4].

While α-Gal A activity testing alone is sufficient to diagnose FD in males, it is necessary to identify a disease-causing mutation in GLA in females, who typically exhibit normal or highly variable plasma α-Gal A activity due to skewed X chromosome inactivation[5]. Routine methods for GLA genotyping include real-time PCR[6], multiplex PCR[7], Sanger sequencing [8,9], next-generation sequencing (NGS)[9,10], and multiple ligation-dependent probe amplification[11], which collectively identify pathogenic variants in over 96% of FD cases[12]. To date, over 1,000 variants of GLA have been registered in the Human Gene Mutation Database (HGMD) Professional[13,14]. These include missense mutations (58.5%), nonsense mutations (16.5%), deletions (13.0%), splicing defects (10.5%); insertions (1.0%), and insertion-deletion (indel) (0.5%) [12]. With advances in gene mutation-detection technologies, previously undetectable intronic variants are increasingly being recognized. Compared with Sanger sequencing, long-read sequencing (LRS) offers superior detection of almost all types of mutations, including deep intronic variants[15].

In 2002, Ishii et al. uncovered IVS4+919G > A, a variant that lies deep within intron 4 of GLA and causes aberrant splicing of GLA mRNA[16]. Since then, intronic variants have emerged as important contributors to the mutational spectrum of FD. In this study, we report two novel deep intronic GLA variants identified by LRS in two unrelated male FD patients. These variants were not detected by classical Sanger sequencing. Bioinformatic analyses supported the pathogenic potential of both variants. These findings highlight the value of LRS in expanding molecular diagnostics when conventional approaches fall short.

2. Case Description

Both presented with clinical, enzymatic, and pathological features suggestive of FD, but Sanger sequencing targeting GLA exons and exon boundaries (National Center for Biotechnology Information reference sequence NM_000169.3) failed to detect pathogenic variants[17]. For further precise the identification and validation of the possible inversion, we performed GLA LRS, RNA Sequencing and quantitative reverse transcription-polymerase chain reaction (RT-qPCR) (Supplementary File S1). Patient data were obtained through retrospective clinical review, and α-Gal A activity and lyso-Gb3 levels were determined using dried blood spot samples.

2.1. Patient 1

Patient 1, a 20-year-old Han Chinese man, presented with hypohidrosis and unexplained acroparesthesia since childhood, triggered by physical exertion, heat, or emotional stress. Additional symptoms included memory decline, occasional palpitations, dizziness, intermittent tinnitus, fatigue, and reduced exercise tolerance. The patient had undergone intussusception surgery at 8 months of age. Family history was negative for FD (Figure 1A). Renal, heart, and brain assessments were unremarkable. Biochemical testing revealed markedly reduced α-Gal A enzyme activity in leukocytes (0.64 µmol/L/h; reference range: 2.40–17.65) and elevated plasma lyso-Gb3 levels (19.84 ng/mL; reference: < 1.11). Skin biopsy showed myeloid bodies in epithelial cells of sweat glands, consistent with FD (Figure 2). Sanger sequencing did not detect pathogenic GLA variants.

LRS identified a ~1.7-kb insertion within intron 4 of GLA, located between g.100654565 and g.100654566 (NC_000023.10). The insertion corresponded to part of a long interspersed nuclear element-1 (LINE-1), annotated using the RepeatMasker web server under default settings (https://www.repeatmasker.org/cgi-bin/WEBRepeatMasker). A 16-bp non-tandem duplication from intron 4 was present at both ends of the insertion. According to Human Genome Variation Society nomenclature[18], this variant is described as NC_000023.10: g.[100654565_100654566ins1.7kb;100654550_100654565dup] (Figure 3A, B; see Supplementary File S2 for the insert sequence). Sanger sequencing confirmed 5′ and 3′ breakpoints (Figure 3C, D), and maternal testing was negative for the insertion.

RNA-seq of the case patient revealed two aberrant GLA transcripts: one exhibited exon 5 skipping (NM_000169.3:r.640_801del), resulting in an in-frame deletion; the other showed intron 3 retention (NM_000169.3:r.547_548ins547+1_547+89), introducing a premature termination codon (NP_000160.1:p.Gly183fsTer28; Figure 3A). The latter transcript was predicted to undergo nonsense-mediated decay.

Splicing analysis of the 1.7-kb insert using the Human Splicing Finder (HSF) web tool (https://hsf.genomnis.com) identified 152 splicing signals, including 85 acceptor and 67 donor splice sites, 1,134 exonic splicing silencer (ESS) sites, 987 exonic splicing enhancer (ESE) sites, and 74 branch point sequences (BPSs). The exonic splicing regulatory (ESR) ratio, calculated as the number of ESEs divided by the number of ESSs, was < 1 (1,134/987) (Figure 3E).

2.2. Patient 2

Patient 2, a 37-year-old Han Chinese man, presented with acroparesthesia and hypohidrosis starting at the age of 8 years. His medical history included hypertension, nephrotic syndrome, and a positive family history of FD. He underwent dialysis for 4 years prior to undergoing a kidney transplant at age 35. During the course of his illness, he also experienced tinnitus, hearing loss, palpitations, and diarrhea. Echocardiography showed left ventricular hypertrophy (left ventricular posterior wall thickness: 1.6 cm). Lab results revealed reduced α-Gal A activity (0.49 µmol/L/h), and elevated lyso-Gb3 levels (52.01 ng/mL). His older brother developed similar symptoms at age 8 and had a history of hypertension, hypertrophic cardiomyopathy, and lacunar infarction; he died after initiating dialysis at age 33. The patient’s 56-year-old mother reported burning sensations in her feet beginning at age 8, which disappeared after childbirth. The patient is currently receiving enzyme replacement therapy (Figure 1B).

LRS identified a 2.5-kb insertion within the 5′-untranslated region (UTR) located between g.100662958 and g.100662959, along with a 21-adenine nucleotide duplication and an 18-bp duplication (g.100662941_100662958) downstream of the insertion. RepeatMasker annotated the inserted sequence as part of a SINE-VNTR-Alu (SVA) retroposon element. Homology analysis using BLAST revealed similarity between the insertion sequence and a deep intronic region of RNASE10 intron 1 (GRCh37 chr14:g.20973426-20975810). This variant is described as NC_000023.10: g.[100662958_100662959ins2.5kb;G [21];100662941_100662958dup] (Figure 4A–D). Sanger sequencing confirmed the variants.

Unlike Patient 1, RNA-seq did not detect aberrant GLA transcripts. However, RT-qPCR analysis showed reduced expression of exons 1, 3, and 7 in normal GLA transcripts compared with healthy controls (Figure 4E).

3. Discussion

We report two novel non-coding GLA variants in two unrelated male patients with FD, including a 1.7-kb LINE-1 insertion within intron 4 associated with abnormal splicing, and a 2.5-kb insertion in the 5′-UTR homologous to RNASE10 intron 1 linked to reduced expression of normal transcripts. Although NGS, which enables analysis of numerous genes and detection of complex variants, has been successfully used in FD diagnosis, deep intronic variants may still be missed because of limitations in exon-focused panels. LRS can identify complex genetic variants, including deep intronic variants and structural rearrangements, complementing routine diagnostic methods. In a cohort study of 207 Japanese patients with FD, seven (3.4%) had no pathogenic variants detected in the exonic regions or exon–intron boundaries of GLA using standard gene analysis[12]. In a study by Nowak et al., two of 12 patients showed no pathogenic variants on Sanger sequencing. Multiple amplicon sequencing subsequently identified a deep intronic variant (c.547+404T>G) in one patient, while mRNA and cDNA sequencing showed an exon 2 deletion in the other[19]. In a 2024 cohort of 82 FD patients, Yao et al. reported three deep intronic variants identified by long-range PCR coupled with LRS that had been missed by Sanger sequencing[15]. Similarly, our study uncovered two insertional variants, one in intron 4 and the other in the 5′-UTR region of GLA, in FD patients who previously tested negative by conventional sequencing. Based on clinical symptoms, α-Gal A activity, and lyso-Gb3 levels, these two patients were classified as having non-classic and classic FD, respectively[17].

Located more than 100 bp away from exon–intron boundaries, these deep intronic variants can affect ESEs or ESSs, potentially causing misregulated splicing[20]. Splicing of pre-mRNAs removes introns and joins exons to form mature mRNA transcripts for translation. This process is guided by signals such as splice sites, BPSs, and regulatory elements, including ESEs and ESSs, which promote or inhibit the inclusion of coding segments in the final mRNA[21,22,23,24]. The prevalent c.639+919G >A mutation disrupts the binding of heterogeneous nuclear ribonucleoprotein A1/A2 to an ESS, causing pseudoexon activation and FD[25]. In our Patient 1, the ESR ratio was < 1, suggesting a predominance of splicing silencers over enhancers, which may contribute to exon skipping or impaired exon recognition during pre-mRNA splicing.

LINE-1 elements can drive inherited diseases through various mechanisms, including de novo LINE-1 insertions, insertion-mediated deletions, and genomic rearrangements[26]. Wimmer et al.[27] reported a patient with a LINE-1 insertion in intron 9 of the NF1 gene, resulting in the skipping of the preceding exon and exonization of a short portion of the LINE-1 sequence. Alesi et al.[28] described two siblings affected by neurofibromatosis type 1, in whom insertion of a 5′-truncated inverted LINE-1 element into intron 15 led to skipping of the upstream exon and generation of an alternative transcript lacking exon 15. Similarly, our Patient 1 was found to harbor a ~1.7-kb insertion derived from a LINE-1 element, predicted to disrupt normal splicing and result in aberrant transcription.

The 5′- and 3′-UTRs are critical for post-transcriptional gene regulation, influencing processes such as mRNA processing, stability, and translation initiation[29]. The 5′-UTR, plays a particularly important role in ribosome recruitment and translation efficiency, impacting cellular proteome expression[30,31]. Variants in the 5′-UTR have been shown to impair translation and lead to disease, as exemplified by the heterozygous deletion of MEN1[32] and point mutation of FGF13[33]. SVA insertions can affect gene expression through mechanisms such as exon trapping and alternative splicing, potentially leading to truncated proteins[34]. Pathogenic SVA insertions have been implicated in various diseases, including X-linked dystonia-parkinsonism (XDP), breast cancer, Fukuyama muscular dystrophy, and Batten disease[34]. XDP cell models exhibit multiple transcriptional abnormalities surrounding the exons flanking the SVA insertion, including aberrant alternative splicing, partial intron retention, decreased transcription of exons, and decreased levels of the full-length TAF1 mRNA[35]. In our Patient 2, we identified an SVA insertion within the GLA 5′-UTR that resulted in reduced gene expression. This discovery represents a crucial step toward understanding the molecular mechanisms by which retrotransposons contribute to FD pathogenesis, a phenomenon not previously reported.

4. Conclusions

Our study broadens the known genetic spectrum of GLA variants and provides further insight into the molecular basis of FD. When conventional Sanger sequencing fails to detect pathogenic variants, LRS should be considered. Integrating LRS into the diagnostic workflow ensures a more comprehensive approach, reducing the risk of missing clinically relevant variants in cases with negative results from conventional testing.