SNaPshot Assay in Quantitative Detection of Allelic Nondisjunction in Down Syndrome.

AIM: We wished to identify markers associated with allelic nondisjunction in nuclear families with Down syndrome (DS) offspring. Since the GRIK1 and GARS-AIRS-GART genes, mapping to chromosome 21q22.1, may be informative in this regard, we genotyped four single-nucleotide polymorphisms [30952599(A/G) rs363484; 30924733(A/G) rs363506; 34901423(A/G) rs2834235; 34877070(A/G) rs7283354] present in these genes using the SNaPshot(™) assay protocol. RESULTS: We have reported 30952599(A/G)-rs363484 to be monomorphic in our sample population. Genotyping revealed 35/65 families to be informative for 34877070(A/G)-rs7283354 (GARS-AIRS-GART), whereas only 25/65 and 11/65 are informative for 34901423(A/G)-rs2834235 (GARS-AIRS-GART) and 30924733(A/G)-rs363506 (GRIK1) polymorphisms, respectively. The parent- and stage-of-origin of nondisjunction could be traced in 48/65 families using at least one polymorphic marker. A single trio provided internal validation for assignment of the parent- and stage-of-origin of nondisjunction whereby the nondisjoining alleles were independently identified as G-rs363506, G-rs2834235, and G-rs7283354, respectively. An enhanced ratio of meiosis-I to meiosis-II errors during maternal or paternal meioses accounts for allelic nondisjunction. CONCLUSIONS: The SNaPshot assay is quantitative and permits multiplexing for detection of allelic nondisjunction. Inclusion of additional informative chromosome 21-specific markers may aid rapid aneuploidy detection, screening, and prenatal counseling of parents at risk of having babies with DS.

No Evidence for Mutations that Deregulate GARS-AIRS-GART Protein Levels in Children with Down Syndrome.

UNLABELLED: GARS-AIRS-GART is crucial in studies of Down syndrome (DS)-related mental retardation due to its chromosomal location (21q22.1), involvement in de novo purine biosynthesis and over-expression in fetal DS brain postmortem samples. GARS-AIRS-GART regions important for structure-function were screened for mutations that might alter protein levels in DS patients. Mutation screening relied on multiplex/singleplex PCR-based amplification of genomic targets followed by amplicon size determination/fingerprinting. Serum protein samples were resolved by SDS-PAGE and immunoblotted with a GARS-AIRS-GART monoclonal antibody. No variation in amplicon size/fingerprints was observed in regions encoding the ATP-binding, active site residues of GARS, the structurally important glycine-rich loops of AIRS, substrate-binding, flexible and folate-binding loops of GART or the poly-adenylation signal sequences. The de novo occurrence or inheritance of large insertion/deletion/rearrangement-type mutations is therefore excluded. Immunoblots show presence of GARS-AIRS-GART protein in all patient samples, with no change in expression levels with respect to either sex or developmental age. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s12291-011-0183-6) contains supplementary material, which is available to authorized users.

Phylogenetic analysis and in silico characterization of the GARS-AIRS-GART gene which codes for a tri-functional enzyme protein involved in de novo purine biosynthesis.

Human GARS-AIRS-GART encodes a fused tri-functional enzyme protein involved in de novo purine biosynthesis, aberrant function being implicated in Down syndrome and Leukemia. We performed phylogenetic analysis to discern evolutionary relationships and in silico characterization to identify elements potentially important for gene regulation. We report that murine, bovine and chimpanzee sequences are the nearest neighbors of human GARS-AIRS-GART and that endo-duplication of the AIRS protein is restricted to insect orthologs. Convergent evolution of mono-functional bacterial orthologs to bi-functional, partly fused, yeast orthologs is observed from the rooted-NJ tree topology that bears bootstrap values exceeding 9000 in majority of the nodes. Sequence alignments reveal that introns 11-15 of human GARS-AIRS-GART are conserved among vertebrates. An inverse correlation is observed between intron size and intron density without bias for intron position. The generation time of organisms is independent of intron density. Human, bovine and murine sequences possess similar GC content with CpG islands in promoter regions. The long isoforms of cow and chicken transcripts and short isoforms of human, bovine and murine mRNA form energetically stable stem-like structures in the 3'-UTR and may regulate translational stability of GARS-AIRS-GART transcripts. Glycine-rich loops important for enzyme structure and ATP-, folate-binding residues are partially conserved.

Potential interaction between the GARS-AIRS-GART Gene and CP2/LBP-1c/LSF transcription factor in Down syndrome-related Alzheimer disease.

(1) GARS-AIRS-GART is an important candidate gene in studies of Down syndrome (DS)-related Alzheimer's disease (AD), due to its chromosomal localization (21q22.1) in the Down syndrome critical region, involvement in de novo purine biosynthesis, and over-expression in DS brain. The aim of this study was to identify factor(s) likely to enhance transcription of GARS-AIRS-GART in DS-related AD. (2) Based on a bio-informatics approach, the PromoterInspector, Promoter Scan II, and EBI toolbox CpG plot software programs were used to identify GARS-AIRS-GART sequences important for gene transcription. Transcription factor binding motifs within these regions were mapped with the help of the MatInspector and TFSEARCH programs. Factors implicated in neurodevelopment or neurodegeneration were the focus of attention, and mining of human (T1Dbase) and murine (GNF) expression databases revealed information on the regional distribution of these factors and their relative abundance vis-a-vis GARS-AIRS-GART. (3) The Leader-binding protein 1-c (LBP-1c/CP2/LSF) emerged as a promising candidate from these studies, as MatInspector and TFSEARCH analyses revealed a total of four CP2 binding sites with potential for functional interaction(s) within the promoter and CpG islands of GARS-AIRS-GART. Furthermore, two of these sites harbor sequences for methylation-sensitive restriction enzymes, which suggest that methylation status may, in part, regulate CP2-mediated transcription of GARS-AIRS-GART. A search of T1Dbase and GNF expression databases reveals co-expression of CP2 and GARS-AIRS-GART in brain regions relevant to DS-related AD. (4) The virtual screen identified CP2/LBP-1c/LSF as a factor that likely mediates enhanced transcription of GARS-AIRS-GART in DS-related AD.