Fabry disease, an X-linked inborn error of glycosphingolipid catabolism, results from mutations in the gene encoding the lysosomal exoglycohydrolase, alpha-galactosidase A (alpha-Gal A; GLA). In two unrelated classically affected males, two alpha-Gal A missense mutations were identified: R112C + D313Y (c.334C>T + c.937G>T) and C172G + D313Y (c.514T>G + c.937G>T). The D313Y lesion was previously identified in classically affected males as the single mutation [Eng et al., 1993] or in cis with another missense mutation, D313Y + G411D (c.937G>T + c.1232G>A) [Guffon et al., 1998]. To determine whether the D313Y mutation was a deleterious mutation or a coding region sequence variant, the frequency of D313Y in normal X-chromosomes, as well as its enzymatic activity and subcellular localization in COS-7 cells was determined. D313Y occurred in 0.45% of 883 normal X-chromosomes, while the R112C, C172G, and G411D missense mutations were not detected in over 500 normal X-chromosomes. Expression of D313Y in COS-7 cells resulted in approximately 60% of wild-type enzymatic activity and showed lysosomal localization, while R112C, C172G, G411D, and the double-mutated constructs had markedly reduced or no detectable activity and were all retained in the endoplasmic reticulum. The expressed D313Y enzyme was stable at lysosomal pH (pH 4.6), while at neutral pH (pH 7.4), it had decreased activity. A molecular homology model of human alpha-Gal A, based on the X-ray crystal structure of chicken alpha-galactosidase B (alpha-Gal B; alpha-N-acetylgalactosaminidase) was generated [Garman et al., 2002], which provided evidence that D313Y did not markedly disrupt the alpha-Gal A enzyme structure. Thus, D313Y is a rare exonic variant with about 60% of wild-type activity in vitro and reduced activity at neutral pH, resulting in low plasma alpha-Gal A activity.

Fabry disease, an X-linked recessive inborn error of glycosphingolipid catabolism, results from the deficient activity of the lysosomal exoglycohydrolase, α-galactosidase A (EC 3.2.1.22; α-Gal A). The molecular lesions in the α-Gal A gene causing the classic phenotype of Fabry disease in 66 unrelated families were determined. In 49 families, 50 new mutations were identified, including: 29 missense mutations (N34K, T41I, D93V, R112S, L166G, G171D, M187T, S201Y, S201F, D234E, W236R, D264Y, M267R, V269M, G271S, G271V, S276G, Q283P, A285P, A285D, M290I, P293T, Q312H, Q321R, G328V, E338K, A348P, E358A, Q386P); nine nonsense mutations (C56X, E79X, K127X, Y151X, Y173X, L177X, W262X, Q306X, E338X); five splicing defects (IVS4-1G > A, IVS5-2A > G, IVS5 + 3A > G, IVS5 + 4A > G, IVS6-1G > C); four small deletions (18delA, 457delGAC, 567delG, 1096delACCAT); one small insertion (996insC); one 3.1 kilobase Alu-Alu deletion (which included exon 2); and one complex mutation (K374R, 1124delGAG). In 18 families, 17 previously reported mutations were identified, with R112C occurring in two families. In two classically affected families, affected males were identified with two mutations: one with two novel mutations, D264Y and V269M and the other with one novel (Q312H) and one previously reported (A143T) mutation. Transient expression of the individual mutations revealed that D264Y and Q312H were localised in the endoplasmic reticulum and had no detectable or markedly reduced activity, whereas V269M and A143T were localised in lysosomes and had approximately 10 per cent and approximately 35 per cent of expressed wild-type activity, respectively. Structural analyses based on the enzyme's three-dimensional structure predicted the effect of the 29 novel missense mutations on the mutant glycoprotein's structure. Of note, three novel mutations (approximately 10 per cent) were predicted not to significantly alter the glycoprotein's structure; however, they were disease causing. These studies further define the molecular heterogeneity of the α-Gal A mutations in classical Fabry disease, permit precise heterozygote detection and prenatal diagnosis, and provide insights into the structural alterations of the mutant enzymes that cause the classic phenotype.

Pharmacogenetic testing is becoming more common; however, very few quality control and other reference materials that cover alleles commonly included in such assays are currently available. To address these needs, the Centers for Disease Control and Prevention's Genetic Testing Reference Material Coordination Program, in collaboration with members of the pharmacogenetic testing community and the Coriell Cell Repositories, have characterized a panel of 107 genomic DNA reference materials for five loci (CYP2D6, CYP2C19, CYP2C9, VKORC1, and UGT1A1) that are commonly included in pharmacogenetic testing panels and proficiency testing surveys. Genomic DNA from publicly available cell lines was sent to volunteer laboratories for genotyping. Each sample was tested in three to six laboratories using a variety of commercially available or laboratory-developed platforms. The results were consistent among laboratories, with differences in allele assignments largely related to the manufacturer's assay design and variable nomenclature, especially for CYP2D6. The alleles included in the assay platforms varied, but most were identified in the set of 107 DNA samples. Nine additional pharmacogenetic loci (CYP4F2, EPHX1, ABCB1, HLAB, KIF6, CYP3A4, CYP3A5, TPMT, and DPD) were also tested. These samples are publicly available from Coriell and will be useful for quality assurance, proficiency testing, test development, and research. Many laboratories are testing for pharmacogenetic (PGx) markers, common genetic variants that are usually considered only when a patient is likely to be exposed to a Accepted for publication June 21, 2010. R.B., A.E.-B., C.S., A.V., and M.Z. are employees of AutoGenomics (manufacturer of several pharmacogenetic assays used in this study); M.B., A.B., and K.M. are employees of Quest Diagnostics Inc.; J.M. is an employee of Idaho Technology (manufacturer of the reagents used to genotype CYP2C9 and VKORC1 loci for this project);

Considerable progress in tomato molecular biology has been made over the past five years. At least 19 different mRNAs which increase in amount during tomato fruit ripening have been cloned and genes for enzymes involved in cell wall degradation (polygalacturonase and pectinesterase) and ethylene synthesis (ACC synthase) have been identified by conventional procedures. Transgenic plants have been used to identify regions of DNA flanking fruit-specific, ripening-related and ethylene-regulated genes and trans-acting factors which bind to these promoters have also been identified. Antisense genes expressed in transgenic plants have proved to be highly effective for inhibiting the specific expression of ripening-related genes. These experiments have changed our understanding of how softening occurs in tomato fruit. Antisense techniques have also been used to identify genes encoding enzymes for carotenoid biosynthesis (phytoene synthase) and ethylene biosynthesis (the ethylene-forming enzyme). The altered characteristics of fruit transformed with specific antisense genes, such as retarded ripening and resistance to splitting, may prove to be of value to fruit growers, processors and ultimately the consumer.