A study of GluK1 kainate receptor polymorphisms in Down syndrome reveals allelic non-disjunction at 1173(C/T).
TL;DR: Evidence for allelic non-disjunction in the GluK1 gene that encodes the critical kainite-binding glutamate receptor subunit-5, maps to chromosome 21q22.1 in the DS critical region and is expressed in brain regions responsible for learning and memory is sought.
Abstract: Mechanisms underlying Down syndrome (DS)-related mental retardation (MR) remain poorly understood. In trisomic offspring, non-disjunction may result in the reduction to homozygosity of a susceptibility allele inherited from a heterozygous parent. Accordingly, we sought evidence for allelic non-disjunction in the GluK1 gene that encodes the critical kainite-binding glutamate receptor subunit-5, maps to chromosome 21q22.1 in the DS critical region and is expressed in brain regions responsible for learning and memory. Three polymorphisms of GluK1 [522(A/C) rs363538; 1173(C/T) rs363430 and 2705(T/C) rs363504] were genotyped in 86 DS patient families by means of PCR-coupled RFLP assays and evaluated with respect to allele frequency, heterozygosity, linkage disequilibrium, stage and parental origin of allelic non-disjunction. We report that the distribution of allele frequencies is in Hardy-Weinberg equilibrium. Moderate heterozygosity (0.339) and a major allele frequency of 0.78 render the 1173(C/T) marker informative. Pair-wise comparisons reveal that 522(A/C)-1173(C/T) [χ2 = 31.2, df = 1, p = 0.0001; D’ = 0.42] and 1173(C/T)-2705(T/C) [χ2 = 18.3, df = 1, p = 0.0001; D’ = 0.34] are in significant linkage disequilibrium of weak magnitude. The estimated ratio of meiosis-I to meiosis-II errors arising from allelic non-disjunction of 1173(C/T) is 4:1 in maternal cases and 2:1 in paternal cases. Studies including additional markers and patient samples are warranted to further substantiate present findings.
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TL;DR: The majority of the genome is unconstrained by natural selection currently, in agreement with what has been estimated from phylogenetic methods but in sharp contrast to estimates based on transcriptomics or other high-throughput functional methods.
Abstract: The comparative genomics revolution of the past decade has enabled the discovery of functional elements in the human genome via sequence comparison. While that is so, an important class of elements, those specific to humans, is entirely missed by searching for sequence conservation across species. Here we present an analysis based on variation data among human genomes that utilizes a supervised machine learning approach for the identification of human-specific purifying selection in the genome. Using only allele frequency information from the complete low-coverage 1000 Genomes Project data set in conjunction with a support vector machine trained from known functional and nonfunctional portions of the genome, we are able to accurately identify portions of the genome constrained by purifying selection. Our method identifies previously known human-specific gains or losses of function and uncovers many novel candidates. Candidate targets for gain and loss of function along the human lineage include numerous putative regulatory regions of genes essential for normal development of the central nervous system, including a significant enrichment of gain of function events near neurotransmitter receptor genes. These results are consistent with regulatory turnover being a key mechanism in the evolution of human-specific characteristics of brain development. Finally, we show that the majority of the genome is unconstrained by natural selection currently, in agreement with what has been estimated from phylogenetic methods but in sharp contrast to estimates based on transcriptomics or other high-throughput functional methods.
27 citations
Cites background from "A study of GluK1 kainate receptor p..."
...This gene has been associated with autism (Haldeman-Englert et al. 2010), Down syndrome (Ghosh et al. 2009), and juvenile absence epilepsy (Sander et al. 1997), and its expression levels are altered in patients with schizophrenia and bipolar disorder (Woo et al. 2007)....
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TL;DR: It is demonstrated that novelty detection per se and the encoding of visuo-spatial information was not disrupted in adult Tc1 mice, and the task specific nature of the shortterm recognition memory deficit suggests that the trisomy of genes on human chromosome 21 in T c1 mice impacts on (perirhinal) cortical systems supporting short-term object and olfactory recognition memory.
24 citations
TL;DR: Data obtained indicate that the rs2073601 ‘A’ allele, responsible for nonsynonymous substitution of leucine to methionine, may have some role in DS in this population of eastern Indian probands.
Abstract: Trisomy of the 21st chromosome leads to an over dosage of several regulatory genes in Down syndrome (DS). Though allelic and genotypic combinations formed between genes are interesting, till date, this particular area has never been explored in DS. In the present investigation four SNPs in two transcription factors, Single minded 2 (SIM2) and V-ets erythroblastosis virus E26 oncogene homolog2 (ETS2), located in the 21st chromosome were genotyped to understand their role in DS. Genomic DNA of eastern Indian probands with DS (N = 132), their parents (N = 209) and ethnically matched controls (N = 149) was subjected to PCR-based analyses of functionally important SNPs followed by statistical analyses. ETS2 rs461155 showed high heterozygosity in DS. Significantly lower frequency of SIM2 C-G haplotype (rs2073601-rs2073416) was noticed in individuals with DS (P value = 0.01669) and their fathers (P value = 0.01185). Significantly lower frequency of the A-C-C-G with higher frequency of A-C-A-G haplotypes was also noticed in subjects with DS (P value = 0.02089 and 0.00588 respectively). Data obtained indicate that the rs2073601 ‘A’ allele, responsible for nonsynonymous substitution of leucine to methionine, may have some role in DS in this population.
5 citations
Cites methods or result from "A study of GluK1 kainate receptor p..."
...As was proposed by previous investigators [25], in case of homozygous proband of two heterozygous parents, the stage of nondisjunction was speculated as meiosis II....
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...Similar observationswere reported for the GluK1 kinate receptor in the eastern Indian population, another gene located at the DSCR [25]....
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...In the present investigation, for the first time haplotypic association of SIM2 and ETS2, two TFs located in the DSCR, have been analyzed in probands with DS....
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TL;DR: In this paper, a supervised machine learning approach was used to identify human specific purifying selection in the human genome using only allele frequency information from the complete low coverage 1000 Genomes Project dataset in conjunction with a support vector machine trained from known functional and non-functional portions of the genome.
Abstract: The comparative genomics revolution of the past decade has enabled the discovery of functional elements in the human genome via sequence comparison. While that is so, an important class of elements, those specific to humans, is entirely missed by searching for sequence conservation across species. Here we present an analysis based on variation data among human genomes that utilizes a supervised machine learning approach for the identification of human specific purifying selection in the genome. Using only allele frequency information from the complete low coverage 1000 Genomes Project dataset in conjunction with a support vector machine trained from known functional and non-functional portions of the genome, we are able to accurately identify portions of the genome constrained by purifying selection. Our method identifies previously known human-specific gains or losses of function and uncovers many novel candidates. Candidate targets for gain and loss of function along the human lineage include numerous putative regulatory regions of genes essential for normal development of the central nervous system, including a significant enrichment of gain of function events near neurotransmitter receptor genes. These results are consistent with regulatory turnover being a key mechanism in the evolution of human-specific characteristics of brain development. Finally, we show that the majority of the genome is unconstrained by natural selection currently, in agreement with what has been estimated from phylogenetic methods but in sharp contrast to estimates based on transcriptomics or other high-throughput functional methods.
4 citations
Dissertation•
01 Jan 2016
TL;DR: The behavioural and biochemical analyses provided evidence that the T c1 mouse model showed a selective deficit in short-term recognition memory while sparing longterm memory for the same type of information, and that a near complete copy of human chromosome 21 in Tc1 mice did not impair place recognition.
Abstract: Down’s syndrome is a complex genetic condition arising from trisomy of chromosome 21; it is characterised by alterations in behaviour and synaptic plasticity, leading to deficits in learning and memory. The Tc1 mouse is a transchromosomic mouse model of trisomy-21 which carries a freely segregating and almost complete human chromosome 21. The main aim of this thesis was to explore recognition memory processes in the Tc1 mouse model of trisomy-21.
The initial goal was to provide insight into the learning and memory changes associated with triplication of genes on human chromosome 21 in mice. The Tc1 mouse had previously been shown to display a deficit in object recognition memory following a delay of 10-min, but not following a 24-h delay. This thesis aimed to confirm
and extend these findings, by testing Tc1 mice on an array of novelty based recognition tasks. This thesis also aimed to explore some of the biological systems underpinning the pattern of learning and memory changes demonstrated by the Tc1 mouse. The expression of c- fos was used as a marker of neuronal activity, allowing for the assessment of regional activity, and in addition, the expression profile of the GluR1 subunit of the AMPA receptor and the GluK1 and GluK5 subunits of the Kainate
receptor were examined. Finally, this thesis investigated the impact of the administration of a novel AMPAkine, drug 9A, on the cognitive phenotype of the Tc1 mouse.
The behavioural and biochemical analyses provided evidence that the Tc1 mouse model showed a selective deficit in short-term recognition memory while sparing longterm memory for the same type of information, and that a near complete copy of human chromosome 21 in Tc1 mice did not impair place recognition. In addition, c-fos
expression studies provided evidence for aberrant perirhinal cortex activity in response to familiarity. Further to this, there was a significant reduction in expression of the GluR1 AMPA receptor subunit in the hippocampus, and a significant reduction of the
GluK5 kainate receptor subunit expression in the perirhinal cortex. Finally, this thesis provides some preliminary evidence that a novel positive allosteric modulator, drug 9a, had a positive cognitive enhancing effect in the Tc1 mouse.
2 citations
References
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TL;DR: ClUSTAL X is a new windows interface for the widely-used progressive multiple sequence alignment program CLUSTAL W, providing an integrated system for performing multiple sequence and profile alignments and analysing the results.
Abstract: CLUSTAL X is a new windows interface for the widely-used progressive multiple sequence alignment program CLUSTAL W. The new system is easy to use, providing an integrated system for performing multiple sequence and profile alignments and analysing the results. CLUSTAL X displays the sequence alignment in a window on the screen. A versatile sequence colouring scheme allows the user to highlight conserved features in the alignment. Pull-down menus provide all the options required for traditional multiple sequence and profile alignment. New features include: the ability to cut-and-paste sequences to change the order of the alignment, selection of a subset of the sequences to be realigned, and selection of a sub-range of the alignment to be realigned and inserted back into the original alignment. Alignment quality analysis can be performed and low-scoring segments or exceptional residues can be highlighted. Quality analysis and realignment of selected residue ranges provide the user with a powerful tool to improve and refine difficult alignments and to trap errors in input sequences. CLUSTAL X has been compiled on SUN Solaris, IRIX5.3 on Silicon Graphics, Digital UNIX on DECstations, Microsoft Windows (32 bit) for PCs, Linux ELF for x86 PCs, and Macintosh PowerMac.
38,522 citations
"A study of GluK1 kainate receptor p..." refers background in this paper
...83 [16] under alignment parameters with pair gap penalties of 10....
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TL;DR: A rapid, safe and inexpensive method was developed to simplify the deprotein-ization procedure that yielded quantities comparable to those obtained from phenol-chloroform extractions, rendering the entire process of RFLP analysis free of toxic materials.
Abstract: One of the obstacles encountered when extracting DNA from a large number of samples is the cumbersome method of deprotein-izing cell digests with the hazardous organic solvents phenol and isochloroform. Several other non-toxic extraction procedures have been published, but require either extensive dialysis (1) or the use of filters (2). A rapid, safe and inexpensive method was developed to simplify the deprotein-ization procedure. This method involves salting out of the cellular proteins by dehydration and precipitation with a saturated NaCl solution. Buffy coats of nucleated cells obtained from anticoagulated blood (ACD or EDTA) were resuspended in 15 ml polypropylene centrifugation tubes with 3 ml of nuclei lysis buffer (10 mM Tris-HCl t 400 mM NaCl and 2 mM Na 2 EDTA, pH 8.2). The cell lysates were digested overnight at 37°C with 0.2 ml of 10Z SDS and 0.5 ml of a protease K solution (1 mg protease K in 1Z SDS and 2 mM Na2EDTA). After digestion was complete, 1 ml of saturated NaCl (approximately 6M) was added to each tube and shaken vigorously for 15 seconds, followed by centrifugation at 2500 rpm for 15 minutes. The precipitated protein pellet was left at the bottom of the tube and the supernatant containing the DNA was transferred to another 15 ml polypropylene tube. Exactly 2 volumes of room temperature absolute ethanol was added and the tubes inverted several times until the DNA precipitated. The precipitated DNA strands were removed with a plastic spatula or pipette and transferred to a 1.5 ml microcentrifuge tube containing 100-200 pi TE buffer (10 mM Tris-HCl, 0.2 mM Na 2 EDTA, pH 7.5). The DNA was allowed to dissolve 2 hours at 37°C before quantitating. The DNA obtained from this simple technique yielded quantities comparable to those obtained from phenol-chloroform extractions. The 260/280 ratios were consistently 1.8-2.0, demonstrating good deproteinization. Restrictions were performed using a number of different enzymes requiring high, medium or low salt concentrations, all resulting in complete restriction. This procedure has been used in our laboratory on several thousand blood samples for parentage, population and forensic studies. This technique is used with our non-isotopic hybridization procedures (3) rendering the entire process of RFLP analysis free of toxic materials.
19,905 citations
"A study of GluK1 kainate receptor p..." refers methods in this paper
...The salting out procedure of Miller et al. 1988 [29] was used to isolate genomic DNA from whole blood lymphocytes....
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...1988 [29] was used to isolate genomic DNA from whole blood lymphocytes....
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TL;DR: During 2004, tens of thousands of Knowledgebase records got manually annotated or updated; the UniProt keyword list got augmented by additional keywords; the documentation of the keywords and are continuously overhauling and standardizing the annotation of post-translational modifications.
Abstract: The Universal Protein Resource (UniProt) provides the scientific community with a single, centralized, authoritative resource for protein sequences and functional information. Formed by uniting the Swiss-Prot, TrEMBL and PIR protein database activities, the UniProt consortium produces three layers of protein sequence databases: the UniProt Archive (UniParc), the UniProt Knowledgebase (UniProt) and the UniProt Reference (UniRef) databases. The UniProt Knowledgebase is a comprehensive, fully classified, richly and accurately annotated protein sequence knowledgebase with extensive cross-references. This centrepiece consists of two sections: UniProt/Swiss-Prot, with fully, manually curated entries; and UniProt/TrEMBL, enriched with automated classification and annotation. During 2004, tens of thousands of Knowledgebase records got manually annotated or updated; we introduced a new comment line topic: TOXIC DOSE to store information on the acute toxicity of a toxin; the UniProt keyword list got augmented by additional keywords; we improved the documentation of the keywords and are continuously overhauling and standardizing the annotation of post-translational modifications. Furthermore, we introduced a new documentation file of the strains and their synonyms. Many new database cross-references were introduced and we started to make use of Digital Object Identifiers. We also achieved in collaboration with the Macromolecular Structure Database group at EBI an improved integration with structural databases by residue level mapping of sequences from the Protein Data Bank entries onto corresponding UniProt entries. For convenient sequence searches we provide the UniRef non-redundant sequence databases. The comprehensive UniParc database stores the complete body of publicly available protein sequence data. The UniProt databases can be accessed online (http://www.uniprot.org) or downloaded in several formats (ftp://ftp.uniprot.org/pub). New releases are published every two weeks.
4,074 citations