Fabry disease: 45 novel mutations in the α-galactosidase A gene causing the classical phenotype

Abstract

The nature of the molecular lesions in the α-galactosidase A (α-Gal A) gene causing Fabry disease was determined in 50 unrelated families with the classic phenotype of this X-linked recessive lysosomal storage disease. Genomic DNA was isolated from affected males or obligate carrier females, and the entire α-Gal A coding region as well as the flanking and intronic sequences were analyzed by PCR amplification and automated sequencing. Forty-five new mutations were identified including 38 single base substitutions (32 missense and four nonsense) and nine gene rearrangements: M1R, M42T, G43D, G43V, H46Y, F50C, L68F, G132R, T141I, Y152X, K168R, G183S, V199M, P205R, Y207S, Q221X, C223R, C223Y, D234Y, G271C, A288P, P293A, R301G, I303N, I317T, E341D, P362L, R363C, R363H, G373D, I384N, T385P, Q396X, E398K, S401X, P409A, g7325insC, g7384del13, g8341delG, g8391del4/ins3, g10511delTAGT, g10704delACAG, g11019insG, g11021insG, and g11048delAGG. In the remaining five Fabry families, four previously reported mutations were detected (W81X, R112C, g11011delTC, and g11050delGAG) of which the R112C substitution was found in two families who were unrelated by haplotyping. These studies further define the heterogeneity of mutations in the α-Gal A gene causing the classical Fabry disease phenotype, and permit precise carrier detection and prenatal diagnosis in these families.

Introduction

Fabry disease is an X-linked inborn error of glycosphingolipid catabolism resulting from the deficient activity of the lysosomal exoglycohydrolase, α-galactosidase A (EC 3.2.1.22; α-Gal A) [1]. Affected individuals progressively accumulate globotrioasylceramide and related glycosphingolipids with terminal α-linked galactosyl moieties in the plasma and in tissue lysosomes throughout the body. In classically affected males who have markedly deficient α-Gal A activity, onset of disease manifestations occurs in childhood or adolescence and is characterized by severe acroparesthesias, angiokeratoma, corneal and lenticular opacities, and hypohidrosis. With advancing age, the progressive glycosphingolipid deposition, particularly in vascular endothelial lysosomes, leads to vascular disease of the heart, kidneys, and brain resulting in early demise, typically in the fourth or fifth decade of life. In contrast to the classical phenotype, variants have been identified who present later in life with cardiac disease (e.g., cardiomegaly, cardiomyopathy), and lack the major classical manifestations [2], [3]. Of note, all cardiac variants reported to date have had missense mutations that encoded mutant enzymes with residual activity (e.g., approximately 1–10% of mean normal activity in cultured cells).

Although the diagnosis of affected males with Fabry disease can be reliably made by demonstrating markedly deficient α-Gal A activity in plasma, leukocytes, or cultured cells [2], the enzymatic identification of carrier females, even obligate heterozygotes, is less reliable due to random X-chromosomal inactivation [1], [3], [4]. In fact, the finding of normal α-Gal A activity in an at-risk female does not exclude heterozygosity [5], [6]. Only the identification of an α-Gal A mutation in an at-risk female will provide precise carrier identification. Thus, mutation and/or haplotype analyses (e.g., [7], [8]) are required for carrier detection, as well as for precise prenatal diagnosis and genotype/phenotype correlations.

The availability of the full-length cDNA and entire 12 kb genomic sequence encoding α-Gal A [9], [10] has facilitated characterization of the mutations causing Fabry disease. To date, a variety of mutations have been identified including missense, nonsense and splice-site mutations, and partial gene rearrangements, including small and large intragenic deletions and insertions (e.g., [8], [11], [12]). Most mutations have been private (i.e., unique to one or a few families), with the exception of certain mutations found in unrelated individuals that occurred at CpG dinucleotides, known hotspots for mutation [13], [14], [15].

Recently, clinical trials have indicated the safety and efficacy of α-Gal A replacement therapy for patients with the classical phenotype [16], [17], [18]. These studies demonstrate the ability of the intravenously administered enzyme to gain access to key sites of pathology and reverse the lysosomal glycosphingolipid accumulation in target sites of pathology, including the skin, kidney, and heart. Therefore, it is important to diagnose affected males and symptomatic carrier females for early therapeutic intervention.

In this communication, the nature of the α-Gal A mutations causing Fabry disease was determined in patients from a series of 50 unrelated families with the classical phenotype. Of note, 90% (45 of 50) of these families had previously undescribed mutations. In the other five families, four previously reported lesions were detected, including one mutation that was found in two unrelated families. The novel mutations included 32 missense and four nonsense mutations, five small deletions, three small insertions, and one complex rearrangement involving a small deletion and insertion.