Maternal age trends support uptake of non-invasive prenatal testing (NIPT) in the low-risk population.

Purpose: To examine trends in patients submitting samples for cell-free DNA screening to determine whether they reflect a shift towards NIPT use in the low-risk population. Methods: A review of demographic information was performed for all specimens submitted to the Ariosa Diagnostics clinical laboratory for the Harmony® prenatal test between January 1, 2014 and December 30, 2017. The proportions of specimens for patients under 35 years and 35 years and older were compared. Results: There was a significant increase in the proportion of specimens submitted by patients under 35, from 47.3% in 2014 to 60.3% in 2017 (Chi-square test, p < .001). Conclusions: The proportion of samples submitted to our laboratory by patients under 35 years has significantly increased in the 4-year subset, which represents the demographics of a diverse group of patients from across the globe. This suggests an increase in uptake of NIPT in the low-risk population.

Characterization of 107 genomic DNA reference materials for CYP2D6, CYP2C19, CYP2C9, VKORC1, and UGT1A1: a GeT-RM and Association for Molecular Pathology collaborative project.

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.

Fabry disease: identification of 50 novel alpha-galactosidase A mutations causing the classic phenotype and three-dimensional structural analysis of 29 missense mutations.

Fabry disease, an X-linked recessive inborn error of glycosphingolipid catabolism, results from the deficient activity of the lysosomal exoglycohydrolase, alpha-galactosidase A (EC 3.2.1.22; alpha-Gal A). The molecular lesions in the alpha-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 alpha-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.

Detection of alpha-galactosidase a mutations causing Fabry disease by denaturing high performance liquid chromatography.

Mutations in the alpha-galactosidase A (alpha-Gal A, GLA) gene cause Fabry disease, an X-linked recessive lysosomal storage disease. The majority of mutations are private, and confirmation of carrier status in females requires the definitive identification of a DNA mutation. In addition, knowledge of a family's mutation enables rapid and precise preimplantation and prenatal genetic testing. Here we report the development and use of DHPLC to rapidly and cost-effectively screen for alpha-Gal A mutations. Optimal DHPLC partial denaturing conditions for mutation detection were established for each PCR amplicon corresponding to the seven alpha-Gal A exons and their adjacent intronic/flanking sequences. At least five known mutations in each exon (45 in total) were screened by DHPLC to validate the method. Mutation detection was then performed in 14 affected males diagnosed by enzyme assay and 39 at-risk females, and the amplicons with abnormal DHPLC profiles were sequenced. In all affected males, and in 32 of the 39 at-risk females, four and 16 previously reported and 10 and 15 new mutations were identified, respectively. Sequencing all seven alpha-Gal A gene amplicons in the seven at-risk females who had normal DHPLC profiles excluded them as mutation carriers. Only one mutation (p.P362L) was not initially identified by its DHPLC profile, but in retrospect the profile was abnormal, emphasizing the need for experience in inspecting the profiles. In addition, this technique detected two new intronic polymorphisms, c.640-16A>G and c.1000-22C>T, with frequencies of 0.14 and 0.25 in both normal individuals and Fabry patients, respectively. This DHPLC method should improve the rapidity and cost-effectiveness of alpha-Gal A mutation identification in affected males and carrier females for Fabry disease.

Fabry disease: characterization of alpha-galactosidase A double mutations and the D313Y plasma enzyme pseudodeficiency allele.

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: novel alpha-galactosidase A 3'-terminal mutations result in multiple transcripts due to aberrant 3'-end formation.

Mutations in the gene that encodes the lysosomal exoglycohydrolase, alpha-galactosidase A (alpha-GalA), cause Fabry disease, an X-linked recessive inborn error of glycosphingolipid catabolism. Human alpha-GalA is one of the rare mammalian genes that has its polyadenylation signal in the coding sequence and lacks a 3' untranslated region (UTR). We identified two novel frameshift mutations, 1277delAA (del2) and 1284delACTT (del4), in unrelated men with classical Fabry disease. Both mutations occurred in the 3' terminus of the coding region and obliterated the termination codon, and del2 also altered the polyadenylation signal. To characterize these mutations, 3' rapid amplification of cDNA ends (RACE) and polymerase chain reactions (PCR) were performed, and the amplicons were subcloned and sequenced. Both mutations generated multiple transcripts with various lengths of 3' terminal sequences, some elongating approximately 1 kb. Mutant transcripts were classified as follows: type I transcripts had terminal in-frame thymidines that created termination codons when polyadenylated, type II had downstream termination codons within the elongated alpha-GalA sequence, and type III, the most abundant, lacked termination codons at their 3' ends. To determine if the type III transcripts were degraded by the recently described cytosolic messenger RNA degradation pathway for messages lacking termination codons, northern blot analysis was performed. However, the finding of similar levels of nuclear and cytoplasmic alpha-GalA mRNA in normal and patient lymphoblasts suggested that mRNA degradation did not result from either mutation. Expression of representative transcript types revealed differences in intracellular localization and/or protein stability and catalytic activity, with most mutant proteins being nonfunctional. Characterization of these 3' mutations identified a novel molecular mechanism causing classical Fabry disease.

Fabry disease: twenty novel alpha-galactosidase A mutations and genotype-phenotype correlations in classical and variant phenotypes.

BACKGROUND: Fabry disease (OMIM 301500) is an X-linked inborn error of glycosphingolipid metabolism resulting from mutations in the alpha-galactosidase A (alpha-Gal A) gene. The disease is phenotypically heterogeneous with classic and variant phenotypes. To assess the molecular heterogeneity, define genotype/phenotype correlations, and for precise carrier identification, the nature of the molecular lesions in the alpha-Gal A gene was determined in 40 unrelated families with Fabry disease. MATERIALS AND METHODS: Genomic DNA was isolated from affected males or obligate carrier females and the entire alpha-Gal A coding region and flanking sequences were amplified by PCR and analyzed by automated sequencing. Haplotype analyses were performed with polymorphisms within and flanking the alpha-Gal A gene. RESULTS: Twenty new mutations were identified (G43R, R49G, M72I, G138E, W236X, L243F, W245X, S247C, D266E, W287C, S297C, N355K, E358G, P409S, g1237del15, g10274insG, g10679insG, g10702delA, g11018insA, g11185-delT), each in a single family. In the remaining 20 Fabry families, 18 previously reported mutations were detected (R49P, D92N, C94Y, R112C [two families], F113S, W162X, G183D, R220X, R227X, R227Q, Q250X, R301X, R301Q, G328R, R342Q, E358K, P409A, g10208delAA [two families]). Haplotype analyses indicated that the families with the R112C or g10208delAA mutations were not related. The proband with the D266E lesion had a severe classic phenotype, having developed renal failure at 15 years. In contrast, the patient with the S247C mutation had a variant phenotype, lacking the classic manifestations and having mild renal involvement at 64 years. CONCLUSIONS: These results further define the heterogeneity of alpha-Gal A mutations causing Fabry disease, permit precise heterozygote detection and prenatal diagnosis in these families, and provide additional genotype/phenotype correlations in this lysosomal storage disease.

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

The nature of the molecular lesions in the alpha-galactosidase A (alpha-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 alpha-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: MIR, 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 alpha-Gal A gene causing the classical Fabry disease phenotype, and permit precise carrier detection and prenatal diagnosis in these families.