The most common cause of fragile X syndrome is expansion of a CGG trinucleotide repeat in the 5'UTR of FMR1. This expansion leads to transcriptional silencing of the gene. However, other mutational mechanisms, such as deletions of FMR1, also cause fragile X syndrome. The result is the same for both the expansion mediated silencing and deletion, absence of the gene product, FMRP. We report here on an 11-year-old boy with a cognitive and behavioral profile with features compatible with, but not specific to, fragile X syndrome. A mosaic deletion of 1,013,395 bp was found using high-density X chromosome microarray analysis followed by sequencing of the deletion breakpoints. We review the literature of FMR1 deletions and present this case in the context of other FMR1 deletions having mental retardation that may or may not have the classic fragile X phenotype.
Classical galactosemia (G/G) is caused by the lack of galactose-1-phosphate uridyltransferase (GALT) activity. A more common clinical variant, Duarte/Classical (D/G) produces partial enzymatic impairment. Although neonatal death due to G/G galactosemia has been largely eliminated by population-based screening and intervention, long-term outcome in some is associated with impaired growth, ovarian failure, dyspraxic speech and neurologic deficits. At least 32 variants in the nucleotide sequence of the GALT gene have been identified and 9 have transferred impaired GALT activity to transformed cells in transfection experiments. We here define the prevalence and biochemical phenotype of two mutations. An A to G transition in exon 6 of the GALT gene converts a predicted glutamine at codon 188 to an arginine (Q188R), and introduces a new HpaII cut site into the gene which enables population screening by polymerase chain reaction. An A to G transition in exon 10 in the GALT gene produces a codon change converting an asparagine to aspartic acid at codon 314 (N314D) and adds an AVA II cut site. We screened a large population for the Q188R and N314D sequence changes to investigate the prevalence of Q188R in G/G galactosemia, the effect of homozygosity for Q188R on outcome, and the prevalence and biochemical phenotype of the N314D sequence change. We found that the Q188R mutation has a prevalence of 62% in a predominately Caucasian population of 107 patients with G/G galactosemia. Homozygosity for Q188R was associated with a poor clinical outcome in a subgroup of these patients. The N314D mutation is associated with the Duarte biochemical phenotype with extraordinary concordance.
Purpose: We previously reported a deletion of the Galactose-1-Phosphate Uridyl Transferase (GALT) gene. This deletion can cause apparent homozygosity for variants located on the opposite allele, potentially resulting in a discrepancy between the biochemical phenotype and the apparent genotype in an individual. The purpose of this study was to determine the deletion breakpoints, allowing the development of a rapid and reliable molecular test for the mutation. Methods: A Polymerase Chain Reaction walking strategy was used to map the 5= and 3= breakpoints. The junction fragment was amplified and sequenced to precisely characterize the deletion breakpoints. Results: The deletion has a bipartite structure involving two large segments of the GALT gene, while Classic galactosemia refers to severe galactose-1-phosphate uridyltransferase (GALT) deficiency and manifests within the first week of life with poor feeding, jaundice, vomiting, liver dysfunction, increased bleeding tendency and septicemia, leading to death if left untreated. 1 The disease can be easily managed by lactose restriction, although some long term complications may not be prevented. Diagnostic work-up for classic galactosemia is often initiated following abnormal newborn screening. Classic galactosemia can be confirmed by extremely low or absent GALT enzymatic activity in the hemolysates; while variant forms or carrier status may require confirmation by further biochemical analysis and/or molecular genotyping of the GALT gene. Our laboratory performs both biochemical phenotyping and molecular genotyping to confirm the diagnosis of galactosemia.The biochemical analysis consists of assessment of GALT enzyme activity, measurement of galactose-1-phosphate (Gal-1-P; the GALT enzyme substrate) and GALT protein isozyme analysis in red cells. The first tier of molecular analysis consists of genotyping for 9 common GALT variants (Q188R, N314D, L218L, S135L, K285N, L195P, T138M, Y209C, and IVS2-2AϾG). These mutations account for the vast majority of galactosemia alleles in various populations. 2 The second tier of testing consists of GALT gene sequencing (the exons and the intron/exon junctions) that will detect less common coding and splice junction mutations. These combined biochemical and molecular approaches provide a comprehensive characterization of the patient's biochemical phenotype and genotype to guide nutritional management and genetic counseling.Our combined approach also enables identification of variants that may otherwise go undetected or lead to erroneous diagnosis. For example, the discrepancy between biochemical phenotype and molecular genotype due to the presence of a deletion on one of the alleles results in apparent homozygosity for the variant on the opposite allele. Previously, a large deletion of about 5 kb in the GALT gene was identified because it resulted in discordance between biochemical phenotype and the apparent genotype. 3,4 We have encountered this mutation numerous times during our testing for galactosemia. Here we describe the ...
The Duarte allele (D) is a missense mutation (N314D) that produces a characteristic isoform and partial impairment of galactose-1-phosphate uridyltransferase (GALT) in human erythrocytes, fibroblasts, and transformed lymphoblasts. The position of this amino acid is distant, however, from presumptive catalytic site(s) as deduced from a three-dimensional model of crystallized Escherichia coli galT protein. To evaluate the mechanism(s) involved in the partial impairment of enzymatic activity, we compared the activity, abundance, biological stability, and mRNA of GALT in human lymphoblastoid cell lines cultured from individuals homozygous for wild-type (WT/WT) and Duarte alleles (N314D/N314D). No other nucleotide differences were present in their GALT genes. The apparent Vmax was reduced in N314D/N314D cells to 31 +/- 3.6 compared to WT/WT of 54 +/- 6.5 nmole UDP-galactose formed/g cell protein/hour. Both genotypes had similar apparent KMs for UDP-glucose of 0.142 +/- 0.057 mM and 0.133 +/- 0.056 mM. This reduced Vmax was associated with a reduced abundance of the 86kD GALT dimer as determined by Western blots and densitometry. Using RNase protection assays, this reduced GALT protein in the N314D/N314D cell lines was not associated with reduced abundance of GALT mRNA. Using cycloheximide (3-[2-(3,5-Dimethyl-2-oxocyclohexyl)-2-hydroxyethyl]glutarimide) inhibition of de novo protein synthesis, GALT enzyme activity, and its dimeric protein had a biological T1/2 of approximately 24 hours in N314D/N314D cell lines as compared to 50 hours for WT/WT lymphoblasts. Upon exposure to 50 degrees C for 15 minutes, N314D/ N314D lymphoblasts retained 45% of GALT activity, whereas controls retained 77% activity. Reduced activity and thermal sensitivity caused by the N314D mutation reverted to control values when a lysine was substituted for a glutamic acid at amino acid 203 in cis (E203K). In summary, N314D/N314D lymphoblasts have reduced GALT enzyme capacity, dimeric protein abundance, biological, and thermal stability. We conclude that the substitution of aspartate for asparagine at amino acid 314 in the human GALT protein reduces the biostability of the active enzyme in human lymphoblasts.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.