Correlation between GLA variants and alpha-Galactosidase A profile in dried blood spot: an observational study in Brazilian patients

Abstract

Background: Fabry disease is a rare X-linked inherited disorder caused by deficiency of α-Galactosidase A. Hundreds of mutations and non-coding haplotypes in the GLA gene have been described; however, many are variants of unknown significance, prompting doubts about the diagnosis and treatment. The α-Galactosidase A enzymatic activity in dried blood spot (DBS) samples are widely used for screening purposes; however, even when values below the normal are found, new tests are required to confirm the diagnosis. Here we describe an analysis of GLA variants and their correlation with DBS α-Galactosidase A enzymatic activity in a large Brazilian population with Fabry disease symptoms. Results: We analyzed GLA variants by DNA sequencing of 803 male patients with suspected Fabry disease or belonging to high-risk populations; in 179 individuals, 58 different exonic variants were detected. From these, 50 are variants described as pathogenic and eight described as variants of unknown significance. The other individuals presented complex non-coding haplotypes or had no variants. Interestingly, the enzymatic activity in DBS was different among pathogenic variants and the other genotypes, including variants of unknown significance; the first presented mean of 12% of residual activity, while the others presented levels above 70% of the activity found in healthy controls. Conclusion: The activity of α-Galactosidase A in DBS was markedly reduced in males with known pathogenic variants when compared with subjects presenting variants of unknown significance, non-coding haplotypes, or without variants, indicating a possible non-pathogenic potential of these latter genotypes. These findings bring a better understanding about the biochemical results of α-Galactosidase A in DBS samples, as well as the possible non-pathogenic potential of non-coding haplotypes and variants of unknown significance in GLA gene. These results certainly will help clinicians to decide about the treatment of patients carrying variants in the gene causing this rare but life-threatening disease.

Accuracy diagnosis improvement of Fabry disease from dried blood spots: Enzyme activity, lyso‐Gb3 accumulation and GLA gene sequencing

“…A previous study suggests that sequencing of the GLA gene is the most useful method for screening for FD, however, they do not detect any patient with the disease. 34 Our results confirm this statement, because in all patients with pathogenic variants the disease was confirmed. Regarding enzyme activity or accumulation of substrate in DBS, the measurement of α-Gal A activity is a better screening method to identify patients with FD in both males and females.…”

“…Similar results were obtained by Varela et al, where they showed a mean enzyme activity in males with pathogenic variants of 0.27 μmol/L/h, much lower than that obtained in men without variants (1.77 μmol/L/h). 34 All the diagnosed patients presented low levels of enzyme activity, but not all showed elevation of lyso-Gb3. Two of the 10 diagnosed males did not present accumulation of lyso-Gb3 (patients #12 and #21), both with a late-onset phenotype and 50 and 72 years old respectively.…”

“…Of them, 23 had the pseudodeficiency allele c.937G > A, that it is known to reduce enzyme activity in vitro but it does not cause FD. 2,34 The other 16 subjects showed intronic variants and complex intronic haplotypes that have been shown that it can cause transcription defects. 35 However, this aspect is not clear yet, so we cannot consider these subjects as FD.…”

The benefits and challenges of family genetic testing in rare genetic diseases—lessons from Fabry disease

“…Although valuable tools to improve diagnosis and identify undiagnosed patients, screening studies for Fabry disease, may reveal individuals with GLA variants of yet unknown significance or non-pathogenic (likely) benign variants (Doheny et al, 2018;Germain, Fouilhoux, et al, 2019;Germain & Jurca-Simina, 2018;Germain et al, 2020;van der Tol et al, 2014;Varela et al, 2020). Reanalysis of the GLA variants identified in 63 previously published screening studies demonstrated that 47.9% of males and 74.1% of females had non-pathogenic variants (Doheny et al, 2018).…”

A Novel Dried Blood Spot Detection Strategy for Characterizing Cardiovascular Diseases

“…Dried blood spot (DBS) technology that can be stored stably enables new possibilities for bioanalytical procedures that can be beneficial for patients, health care providers, and laboratories, and the technology can be inexpensive (8). The sampling can be performed in a non-hospital environment and is suitable for large-scale disease screening (9). DBS technology also reduces the sample processing burden and is characterized by straightforward waste disposal.…”