Six novel mutations in the alpha-galactosidase A gene with classical phenotype. K. Azibi^1,2, C. Caillaud^1,2, J. Manicom¹, J.P. Puech¹, A. Kahn², L. Poenaru^1,2. 1) Biochimie Genetique, Faculte de Medacine Cochin, Paris, France; 2) INSERM U-129, CHU Cochin, Paris, France.
   Fabrys disease is an X-linked glycosphingolipid storage disease resulting from deficient activity of the lysosomal alpha-galactosidase A (GLA). This deficiency leads to the accumulation of uncleaved glycosphingolipids in the lysosomes of vascular endothelial and smooth muscle cells and also in plasma. The clinical manifestations in classical form are due to a small vessels pathology resulting in angiokeratoma, renal failure and heart and brain ischemia. Death occurs from renal or cardiac complications. There are also atypical forms with residual GLA activity and late-onset cardiomyopathy. Patients studied have a classical Fabry disease, with angiokeratoma, abdominal pain, corneal opacity, paresthesias in the extremities and renal failure. One patient presents a dementia. In one family with three patients, one was a female with a polycystic kidney. The alpha-galactosidase was deficient in all these patients with a residual activity between 0,8 to 4 nmol/h/mg protein. To clarify the molecular mechanism causing the enzyme defect and to facilitate a rapid detection of hemizygote and carrier, we used a direct sequencing strategy to determine the GLA gene alterations. The seven exons including intron-exon junctions were PCR amplified and the products directly sequenced. Six novel mutations were identified, including two nonsense mutations (W245X and W262X) and two missense mutations : A156N (G-->C transition generating a novel SfaNI restriction site) and Q279H (C-->A transition creating a novel HphI site). A small nucleotide insertion (InsG10678) was also detected, generating a codon stop at 332 on the cDNA, as well as a small deletion (1235del15) which creates a novel Sau96I restriction site and obliterates a HaeIII site. The affected female carries the novel 1235del15 in the GLA gene. Her clinical expression is probably due to the extreme lyonisation of the normal X-chromosome. Our study further defines the previously reported heterogeneity of mutations causing the disease. It has also permitted to clarify the carrier status and to facilitate the prenatal diagnosis.