Fabry disease: D313Y is an α-galactosidase A sequence variant that causes pseudodeficient activity in plasma
Introduction
Fabry disease is an X-linked recessive lysosomal storage disorder resulting from the deficiency of the exogalactosidase, α-galactosidase A (α-Gal A; EC 3.2.1.22). The enzyme defect leads to the deposition of neutral glycosphingolipids with terminal α-galactosyl residues, predominantly globotriaosylceramide (GL-3), in the plasma and cellular lysosomes throughout the body [1]. In affected males with the classic phenotype, the early clinical features include acroparesthesia, angiokeratoma, hypohidrosis, and corneal and lenticular opacities. With advancing age, the progressive GL-3 deposition, particularly in vascular endothelial cells in the kidneys, heart, and brain, leads to death in the 4th and 5th decades of life due to renal failure, myocardial infarction or heart failure or stroke [1]. Heterozygous females for the classic disease variably express the clinical phenotype. They can be asymptomatic, mildly affected, or have manifestations as severe as those in affected males [1], [2].
In addition to the classical phenotype, milder variants with residual α-Gal A activity have been described who lack the classic features (i.e., acroparesthesia, angiokeratoma, hypohidrosis, and corneal and lenticular opacities). “Cardiac” and “renal” variants present with either late-onset manifestations primarily limited to the heart [3], [4], [5], [6] or kidney (renal variants) [7], respectively.
The gene encoding human α-Gal A is located at Xq22.1 [1]. The full-length ∼1.4 kb cDNA and the entire ∼12 kb genomic sequence, which contains seven exons, have been isolated and characterized [8], [9]. The cDNA encodes a polypeptide of 429 amino acids including a 31-residue leader sequence. The α-Gal A enzyme is glycosylated and functions as a homodimer. To date, over 270 α-Gal A mutations causing Fabry disease have been described of which 160 are missense mutations (Human Gene Mutation Database; www.uwcm.ac.uk/uwcm/mg/hgmd0.html). Several polymorphisms in the gene have been described including −30G→A, −12G→A, −10C→T in the exon 1 promoter region, 7186del7 in intron 2, and the SacI site in intron 4 [10], [11], [12], [13], but no coding region sequence variants have been reported.
In this communication, we report an asymptomatic adult female with low plasma and leukocyte α-Gal A activities who was discovered in the course of routine diagnostic enzyme testing. Sequencing analysis revealed that she had only the D313Y missense mutation. Family studies revealed other relatives including a 30-year-old male who had the D313Y allele and no stigmata of Fabry disease. Previously, the D313Y mutation was reported in classically affected males with Fabry disease either as the single mutation [14] or on the same allele with a second missense mutation, G411D [15]. Therefore, studies were conducted to determine if the D313Y and G411D mutations were disease-causing either alone or in combination. Constructs with D313Y, G411D, and D313Y/G411D were expressed in COS-7 cells to evaluate the activity, biosynthesis, and processing of the encoded polypeptides. The D313Y allele had ∼75% of expressed wild-type activity, whereas the G411D allele and the complex allele D313Y plus G411D (D313Y/G411D) had <3 and <2% of expressed mean wild-type activity, respectively. Biosynthetic studies demonstrated that the D313Y allele encoded a mature enzyme subunit, while the G411D and D313Y/G411D alleles did not. These studies demonstrated that D313Y was a coding sequence variant with about 75% of normal intracellular activity, but a pseudodeficient activity in plasma.
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Probands
Proband 1 was diagnosed at 33 years of age and had the classical phenotype of Fabry disease. Proband 2 was a 50-year-old asymptomatic female with none of the manifestations that occur in affected males or carrier females with Fabry disease, including the absence of corneal and lenticular changes, left ventricular hypertrophy or other cardiac findings, or proteinuria. She had been a normal control for diagnostic enzyme testing and was found to have decreased plasma and leukocyte α-Gal A
Enzyme and substrate determinations
The α-Gal A activities in leukocytes and plasma (Table 2) and the levels of urinary sediment glycosphingolipids were determined in Proband 1, an affected male with classic Fabry disease, and Proband 2, an asymptomatic normal adult female. Proband 1 had essentially undetectable α-Gal A activity in leukocytes, while his activity in plasma was 1.4 nmol/h/ml or about 15% of mean normal activity. Proband 2 had ∼40% of normal mean α-Gal A activity in leukocytes, and ∼20% of normal mean activity in
Discussion
The identification of a normal female with low α-Gal A activity in plasma and leukocytes lead to sequencing her α-Gal A alleles. The finding of the D313Y mutation suggested that she was a heterozygote for Fabry disease. Support for heterozygosity was based on the previous report of a classically affected male with Fabry disease who had the D313Y mutation [14]. However, the absence of a family history and the absence of clinical and biochemical findings for Fabry disease in the normal female or
Acknowledgements
This work was supported by grants from VML (Vaincre les Maladies Lysosomales). We thank, V. Bonnet for technical assistance and M. de Montfalcon for technical help with the glycolipid analysis. This work was supported in part by grants (to R.J.D.) from the National Institutes of Health, including a research Merit Award (5 R37 DK34045), a Grant (5 M01 RR00071) for the Mount Sinai General Clinical Research Center, and a Grant (5 P30 HD28822) for the Mount Sinai Child Health Research Center.
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