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PubMed2026

Genomic Structural Equation Modeling Identifies a Shared Inflammatory Genetic Dimension Across Inflammatory Arthritis Phenotypes and Biomarkers.

BACKGROUND: Inflammatory arthritis (IA), including rheumatoid arthritis (RA), psoriatic arthritis (PsA) and gout, shares systemic inflammatory features indexed by C-reactive protein (CRP) and interleukin-6 (IL-6), yet the extent of their common genetic basis remains unclear. AIMS: We aimed to delineate the shared genetic architecture across IA phenotypes and inflammatory biomarkers. MATERIALS AND METHODS: We applied genomic structural equation modelling (Genomic SEM) to GWAS summary statistics for RA, PsA, gout, CRP and IL-6, fitted a single common factor, and performed multivariate GWAS followed by fine-mapping, transcriptome-wide association, gene-based analysis, pathway enrichment, and cell-type and spatial mapping. RESULTS: A single common factor was fitted (CFI = 0.990, SRMR = 0.045). The multivariate GWAS identified 56 genome-wide significant SNPs across 10 independent lead loci, including one novel signal. Fine-mapping prioritized high-confidence variants near PTPN22, the CRP gene cluster and a urate-associated locus. Gene-level analyses converged on DCLRE1B, PTPN22, IL6R, NLRP3 and HNF1A, with pathway enrichment implicating inflammasome assembly and metabolic-inflammatory overlap. Cell-type enrichment highlighted myeloid populations, and spatial mapping localized signals to lung, kidney, mucosal epithelium and gastrointestinal tissues. DISCUSSION: These results delineate a shared inflammatory genetic dimension across IA phenotypes and biomarkers, anchored in immune, inflammasome, cytokine-receptor and metabolic pathways. CONCLUSION: Together, these findings provide a valuable framework for prioritizing candidate genes and cellular contexts for future investigation.

TWAS↗

PubMed2004

Expression of human proteins at the Southeast Collaboratory for Structural Genomics.

The human protein production group at the Southeast Collaboratory for Structural Genomics is charged with producing human proteins for both X-ray crystallography and NMR structural studies. Eukaryotic, and human proteins in particular, are notoriously difficult to express in bacterial systems. For various reasons, T7-based expression often results in protein expressed in an insoluble form. Overcoming this requires either introduction of a step to screen expression conditions or inclusion of a troublesome refolding step during purification. Our laboratory uses a trc-based expression vector that addresses many of the difficulties of the commonly used T7-based expression systems. Proteins expressed under the trc promoter, a weak promoter compared to the strong T7 promoter, are produced in a soluble form and include necessary cofactors. The details of this system will be discussed.

Cloning, Molecular↗

PubMed2002

SUPFAM--a database of potential protein superfamily relationships derived by comparing sequence-based and structure-based families: implications for structural genomics and function annotation in genomes.

Members of a superfamily of proteins could result from divergent evolution of homologues with insignificant similarity in the amino acid sequences. A superfamily relationship is detected commonly after the three-dimensional structures of the proteins are determined using X-ray analysis or NMR. The SUPFAM database described here relates two homologous protein families in a multiple sequence alignment database of either known or unknown structure. The present release (1.1), which is the first version of the SUPFAM database, has been derived by analysing Pfam, which is one of the commonly used databases of multiple sequence alignments of homologous proteins. The first step in establishing SUPFAM is to relate Pfam families with the families in PALI, which is an alignment database of homologous proteins of known structure that is derived largely from SCOP. The second step involves relating Pfam families which could not be associated reliably with a protein superfamily of known structure. The profile matching procedure, IMPALA, has been used in these steps. The first step resulted in identification of 1280 Pfam families (out of 2697, i.e. 47%) which are related, either by close homologous connection to a SCOP family or by distant relationship to a SCOP family, potentially forming new superfamily connections. Using the profiles of 1417 Pfam families with apparently no structural information, an all-against-all comparison involving a sequence-profile match using IMPALA resulted in clustering of 67 homologous protein families of Pfam into 28 potential new superfamilies. Expansion of groups of related proteins of yet unknown structural information, as proposed in SUPFAM, should help in identifying 'priority proteins' for structure determination in structural genomics initiatives to expand the coverage of structural information in the protein sequence space. For example, we could assign 858 distinct Pfam domains in 2203 of the gene products in the genome of Mycobacterium tubercolosis. Fifty-one of these Pfam families of unknown structure could be clustered into 17 potentially new superfamilies forming good targets for structural genomics. SUPFAM database can be accessed at http://pauling.mbu.iisc.ernet.in/~supfam.

Animals↗

PubMed2000

Genomic structure of mouse IA-2: comparison with its human homologue.

AIMS/HYPOTHESIS: IA-2 is a transmembrane protein with a tyrosine phosphatase (PTP)-like structure and a major autoantigen in Type I (insulin-dependent) diabetes mellitus. Because the nucleotide sequence of human and mouse IA-2 cDNA are closely related, it seemed likely that the genomic organization of the two molecules would be similar. To test this possibility the current experiments were initiated to characterize and compare the genomic structure of mouse and human IA-2. METHODS: IA-2 cDNA was used to screen a 129SVJ mouse genomic library. We selected and mapped 7 overlapping clones. The subcloned inserts were used to determine intron-exon junctions by direct sequencing. Polymerase chain reaction and restriction mapping were used to estimate the size of the introns. RESULTS: The mouse IA-2 gene and the 5' upstream regulatory region were isolated and the intron-exon junctions determined. Mouse IA-2 encompasses approximately 20 kb and encodes 23 exons. Both the 3' and 5' ends were mapped by rapid amplification of cDNA ends (RACE) and a 2 kb 5'-upstream region was shown to have functional promoter activity. CONCLUSION/INTERPRETATION: Comparison of the genomic structure of mouse and human IA-2 shows that they have the same number of exons and nearly identical intron-exon junctions. The region around the major transcription start site of mouse IA-2 is similar to human IA-2 and other transmembrane protein tyrosine phosphatases. It is concluded that human and mouse IA-2 are highly conserved and derived from a common ancestral gene.

Amino Acid Sequence↗

PubMed1999

Fine mapping and genomic structure of ACTN2, the human gene coding for the sarcomeric isoform of alpha-actinin-2, expressed in skeletal and cardiac muscle.

The present paper reports on the fine mapping of the ACTN2 gene and on the reconstruction of its genomic structure. By radiation hybrid mapping, the gene was located about 912 cR from the 1p-telomere. ACTN2 was placed between the marker WI-9317 (alias D1S2421) and the marker AFMA045ZC5, within the chromosomal band 1q43. The gene was detected in YAC 955 c 12. This YAC was used as template DNA for long-distance and Alu-PCR, using a set of putative exonic primers, designed on the cDNA sequence of alpha-actinin-2, in order to characterize the ACTN2 intron-exon boundaries. The entire genomic structure of the gene was reconstructed. The ACTN2 gene contained 21 exons, in a segment spanning about 40 kb of genomic DNA. Only the proximal part of the gene shows a high conservation through evolution, whereas in the remaining part a divergence from the genomic organization of C. elegans and D. melanogaster was noticed. A series of intronic primers was specifically designed and produced, to amplify all the exons of ACTN2, directly from genomic DNA. This will enable mutation screening in patients affected with hereditary diseases linked to the marker CA4F/R, a polymorphism in the last intron of the alpha-actinin-2 gene.

Actinin↗

PubMed1999

Genomic structure and chromosome mapping of the genes encoding clathrin-associated adaptor medium chains mu1A (Ap1m1) and mu1B (Ap1m2).

The protein mu1B is a member of the medium chain family of the clathrin-associated adaptor complex and is expressed exclusively in epithelial cells. We determined the genomic structure of previously cloned murine genes for mu1B (Ap1m2) and its closely related homolog, mu1A (Ap1m1). Comparison of their genomic structures revealed that the positions of introns are identical between these two genes, except for the insertion of an additional intron in Ap1m1 (intron 4). By contrast, these structures are different from that of the more distantly related Ap2m1 gene encoding mu2. Taken together with the similarity of amino acid sequences among these genes, the data presented in this study suggest that Ap1m1/2 and Ap2m1 diverged long before the separation of Ap1m1 and Ap1m2, which most likely resulted from a relatively recent gene duplication. We also mapped AP1M2 to human chromosome 19p13.2 and Ap1m2 to the proximal region of mouse chromosome 9. The results are consistent with the fact that these regions are syntenic.

Adaptor Protein Complex 1↗

PubMed2002

Genomic structure and evolutionary context of the human feline leukemia virus subgroup C receptor (hFLVCR) gene: evidence for block duplications and de novo gene formation within duplicons of the hFLVCR locus.

In this paper we sought to analyze the genomic structure and context of human feline leukemia virus subgroup C receptor (hFLVCR), a human glucarate transporter-like gene at chromosome 1q31, and compare it to that of a paralog (FLVCR14q) at chromosome 14q24. Splicing, polyadenylation, and expression patterns, as estimated by in silico analysis, differed between the two FLVCR genes despite their similar genomic structures, suggesting active and independent evolution of transcriptional and messenger RNA processing patterns after gene duplication. Promoter activity was bi-directional for hFLVCR, but not for its 14q paralog. The upstream 1q transcribed sequences were determined to comprise a novel gene of unknown function, LQK1. Annotation of contigs centered at hFLVCR and FLVCRL14q also revealed highly conserved gene clusters on chromosomes 1 and 14, inferred to result from a duplication. The clusters contained members of the FLVCR, Angel (KIAA0759), JDP, p21SNFT, and TGF- families, as well as two uncharacterized families. The genome-wide locations of both previously recognized and four de novo in silico predicted genes belonging to these seven families were determined. Phylogenetic analyses of these families were consistent with the hypothesis that the 1q/14q duplication occurred early within, or immediately prior to the vertebrate divergence, after the protostome-deuterostome divergence but before the amniote-amphibian divergence.

3' Untranslated Regions↗

PubMed2000

Structural genomics for science and society.

The field of robotics is affecting structural biology, enabling the era of structural genomics. The potential impact on protein fold prediction, biology, protein engineering and medicine is immense. Unraveling mysteries in the protein structure universe will require a dedicated effort for decades to come with computational toxicology as possibly a century long challenge.

Cell Physiological Phenomena↗

PubMed2000

Genomic structure of the human unconventional myosin VI gene.

Mutations in myosin VI (Myo6) cause deafness and vestibular dysfunction in Snell's waltzer mice. Mutations in two other unconventional myosins cause deafness in both humans and mice, making myosin VI an attractive candidate for human deafness. In this report, we refined the map position of human myosin VI (MYO6) by radiation hybrid mapping and characterized the genomic structure of myosin VI. Human myosin VI is composed of 32 coding exons, spanning a genomic region of approximately 70 kb. Exon 30, containing a putative CKII site, was found to be alternatively spliced and appears only in fetal and adult human brain. D6S280 and D6S284 flank the myosin VI gene and were used to screen hearing impaired sib pairs for concordance with the polymorphic markers. No disease-associated mutations were identified in twenty-five families screened for myosin VI mutations by SSCP analysis. Three coding single nucleotide polymorphisms (cSNPs) were identified in myosin VI that did not alter the amino acid sequence. Myosin VI mutations may be rare in the human deaf population or alternatively, may be found in a population not yet examined. The determination of the MYO6 genomic structure will enable screening of individuals with non-syndromic deafness, Usher's syndrome, or retinopathies associated with human chromosome 6q for mutations in this unconventional myosin.

Adult↗

PubMed1998

Molecular diagnosis of McArdle disease: revised genomic structure of the myophosphorylase gene and identification of a novel mutation.

McArdle disease is a rare autosomal recessive disorder of the muscle glycogen metabolism caused by mutations in the muscle glycogen phosphorylase gene. Until now, a total number of 11 different mutations in the coding region or splice sites of the myophosphorylase gene have been identified. In contrast to a wealth of data on the RNA and protein level, little information is available on the genomic sequence of the corresponding gene. To facilitate molecular diagnosis of McArdle disease, we reinvestigated the genomic structure of the myophosphorylase gene and sequenced about 9.8 kilobases (kb) on the genomic level. By choosing 14 intronic primer pairs, we were able to amplify the complete human coding sequence as well as the adjacent splice sites of the 20 exons. Direct sequencing of the amplification products of a consanguineous Turkish family with typical McArdle disease revealed a novel single base pair deletion in exon 18, which predicts a frameshift and a premature termination of the protein. In summary, we established a system for molecular diagnosis of McArdle disease based on a revised genomic structure of the myophosphorylase gene and demonstrated its feasibility by identification of a novel mutation.

Adult↗

PubMed2001

Genomic structure and promoter characterization of an imprinted tumor suppressor gene ARHI.

We have recently identified a maternally imprinted tumor suppressor gene, ARHI (aplysia ras homolog I), the expression of which is lost in ovarian and breast cancers. We have now characterized the genomic structure of the gene including its promoter and the methylation status of its upstream CpG islands. The ARHI gene spans approximately 8 kb containing two exons and one intron. Exon 1 contains 81 non-translated nucleotides, connected to exon 2 with a 3.2-kb intron. The entire protein-coding region is located within exon 2 and encodes a 229-residue small GTP-binding protein belonging to the Ras superfamily. Genomic structure analysis has identified three potential CpG islands. Two of them (CpG island I and II) are located within the promoter and adjacent exon 1 of the ARHI gene. Aberrant methylation of these CpG islands has been detected in breast cancer cells but not in normal epithelial cells, supporting the possibility that appropriate methylation status of the CpG islands in the promoter region may play a role in the downregulation of ARHI gene expression. A TATA box is found 27 bp upstream of the transcription start site associated with several putative transcription factor binding sites. Transient transfection with nested deletion constructs of the 2-kb ARHI promoter regions fused to a luciferase reporter indicated a 121-bp sequence upstream of the transcription initiation site is required for basal promoter activity. Interestingly, this is the region where lower promoter activity has been observed in cancer cells than in normal cells.

Amino Acid Sequence↗

PubMed1997

Production of biologically active recombinant tilapia insulin-like growth factor-II polypeptides in Escherichia coli cells and characterization of the genomic structure of the coding region.

Insulin-like growth factor-II (IGF-II) is a fetal growth factor in humans, but has not been clearly identified in fish up to now. For a detailed understanding of the physiological response of fish IGF-II, the first step was to clone tilapia IGF-II cDNA from the brain cDNA library, coding the region of genomic DNA, and also expressing tilapia IGF-II polypeptides from Escherichia coli. Tilapia cDNA sequences total 1,977 bp, and predicted nucleotide sequences and amino acid sequences of tilapia share 77.9% and 90.7% homology identity with rainbow trout IGF-II, respectively. The genomic structure of the tilapia prepro-IGF-II coding region is very difficult to sequence in mammals and birds. The cloned tilapia IGF-II gene coding region appears much more complex than in other vertebrates. In tilapia IGF-II, the first coding exon I encoding part of the signal peptide sequence is 25 amino acids shorter than the first coding exon of mammals and birds. The other 23 amino acids of the signal peptide, and the first amino acids of the B domain and C domain are encoded by tilapia coding exon 2. The C, A, and D domains, and the first 20 amino acids of the E peptide are encoded by tilapia coding exon 3. The other E peptides and the 3' untranslated region (UTR) region are encoded by tilapia coding exon 4. These data show that the IGF-II genes have significantly differing structures in vertebrate evolution, and there are differences of interrupting introns in the IGF-I genomic structure compared with mammals. To obtain recombinant biologically active polypeptides, tilapia IGF-II B-C-A-D domains were amplified using the polymerase chain reaction (PCR), then ligated with glutathione S-transferase (GST, pGEX-2T vector). Tilapia recombinant IGF-II protein was purified and characterized in E. coli. The fusion protein was also digested with thrombin and appeared as a recombinant IGF-II polypeptide single band with a molecular mass of 7 kD. The recombinant tilapia IGF-II protein biological function was measured by stimulation of [3H]thymidine incorporation. The assay concentration was set up from 0 to 120 nM to stimulate tilapia ovary cell line (TO-2) significantly to uptake thymidine. The results suggest that the recombinant IGF-II protein was dose dependent.

Amino Acid Sequence↗

PubMed1997

Expression, genomic structure and high resolution mapping to 19p13.2 of the human smooth muscle cell calponin gene.

Smooth muscle cells (SMC) express a battery of cell-restricted differentiation genes, many of which are down-regulated during the course of vascular disease. Here, we present the mRNA expression, genomic structure and chromosomal mapping of the gene encoding human smooth muscle cell calponin (SMCC). Human SMCC transcripts are restricted to tissues and cells of SMC origin and, in the latter case, appear to be uniquely controlled in two distinct human SMC lines of uterine and aortic origin. Restriction mapping. Southern blot and PCR analysis of a 70-kb human bacterial artificial chromosome (BAC) revealed a genomic structure (seven exons spanning > 11 kb) very similar to that reported for the mouse SMCC gene. Using a variety of human-rodent somatic cell hybrid and radiation hybrid mapping panels, the human SMCC gene was mapped to a genomic interval of less than 1.32 Mb in 19p13.2. These results provide new information concerning the regulation of SMCC gene expression and demonstrate the utility of two human SMC lines for the further characterization of this gene's expression control. The identification of a BAC harboring the entire human SMCC locus represents an important reagent for future analysis of SMCC regulatory sequences. Finally, the localization of SMCC to a defined genomic interval will facilitate an analysis of its potential as a candidate gene for disease phenotypes mapping to 19p13.2.

Amino Acid Sequence↗

PubMed2002

Target selection for structural genomics: a single genome approach.

We describe our strategy for selecting targets for protein structure determination in context of structural genomics of a single genome. In the course of target selection, we have studied two of the smallest microbial genomes, Mycoplasma genitalium and Mycoplasma pneumoniae. To our surprise, we found that only 71 Mycoplasma genes or their orthologues can be considered as easy targets for high-throughput structural studies--far fewer than expected. We discuss the methods and criteria used for target selection and the reasons explaining rarity of easy targets. First, despite the common opinion that protein folds can be predicted for only 30-50% of genes, the number of "truly unknown" structures is less than one-third. Second, due to the different codon usage, two thirds of Mycoplasma proteins cannot be directly expressed in E. coli in high-throughput manner and require substitution by their homologues from other organisms. Third, membrane or large multi-domain proteins are difficult targets because of solubility and size issues and often require identification and structure determination of protein domains. Finally, we propose different approaches to address the difficult targets.

Cell Membrane↗

PubMed2001

Structural genomics of enzymes involved in sterol/isoprenoid biosynthesis.

X-ray structures of two enzymes in the sterol/isoprenoid biosynthesis pathway have been determined in a structural genomics pilot study. Mevalonate-5-diphosphate decarboxylase (MDD) is a single-domain alpha/beta protein that catalyzes the last of three sequential ATP-dependent reactions which convert mevalonate to isopentenyl diphosphate. Isopentenyl disphosphate isomerase (IDI) is an alpha/beta metalloenzyme that catalyzes interconversion of isopentenyl diphosphate and dimethylallyl diphosphate, which condense in the next step toward synthesis of sterols and a host of natural products. Homology modeling of related proteins and comparisons of the MDD and IDI structures with two other experimentally determined structures have shown that MDD is a member of the GHMP superfamily of small-molecule kinases and IDI is similar to the nudix hydrolases, which act on nucleotide diphosphatecontaining substrates. Structural models were produced for 379 proteins, encompassing a substantial fraction of both protein superfamilies. All three enzymes responsible for synthesis of isopentenyl diphosphate from mevalonate (mevalonate kinase, phosphomevalonate kinase, and MDD) share the same fold, catalyze phosphorylation of chemically similar substrates (MDD decarboxylation involves phosphorylation of mevalonate diphosphate), and seem to have evolved from a common ancestor. These structures and the structural models derived from them provide a framework for interpreting biochemical function and evolutionary relationships.

Amino Acid Sequence↗

PubMed1993

The utrophin and dystrophin genes share similarities in genomic structure.

Utrophin and dystrophin are highly homologous proteins which are reciprocally expressed in DMD (Duchenne muscular dystrophy) muscle. The remarkable similarity of these proteins suggests that they may play a similar cellular role in some circumstances; if this were the case then utrophin may be capable of replacing dystrophin in DMD patients. In this paper we show that the genomic structure of the utrophin gene is similar to the dystrophin gene, further exemplifying the relatedness of the two genes and their gene products. We have constructed a 1.25 Mb contig of eight yeast artificial chromosome (YAC) clones covering the utrophin gene located on chromosome 6q24. Utrophin is encoded by multiple small exons spanning approximately 900 kb. The distribution of exons within the genomic DNA has similarities to that of the dystrophin gene. In contrast to dystrophin, the utrophin gene has a long 5' untranslated region composed of two exons and a cluster of unmethylated, rare-cutting restriction enzyme sites at the 5' end of the gene. Similarities between the genomic structure suggest that utrophin and dystrophin arose through an ancient duplication event involving a large region of genomic DNA.

Base Sequence↗

PubMed1992

The genomic structure of the human skeletal muscle sodium channel gene.

Electrical excitability of neurons and muscle cells reflects the actions of a family of structurally related sodium channels. Mutations in the adult skeletal muscle sodium channel have been associated with the inherited neuromuscular disorders paramyotonia congenita (PMC) and hyperkalemic periodic paralysis (HPP). We have deciphered the entire genomic structure of the human skeletal muscle sodium channel gene and developed a restriction map of the locus. SCN4A consists of 24 exons spanning 35 kb of distance on chromosome 17q. We describe the sequence of all intron/exon boundaries, the presence of several polymorphisms in the coding sequence, and the locations within introns of two dinucleotide repeat polymorphisms. This is the first sodium channel for which the entire genomic structure has been resolved. The organization of the SCN4A exons relative to the proposed protein structure is presented and represents a foundation for functional and evolutionary comparisons of sodium channels. Knowledge of the exon structure and flanking intron sequences for SCN4A will permit a systematic search for mutations in PMC and HPP.

Amino Acid Sequence↗

PubMed1998

Genomic structure of human anion exchanger 3 and its potential role in hereditary neurological disease.

Alterations in ion channel permeability or selectivity have been shown to cause neurological defects in humans. Anion exchanger isoform 3 (AE3) is prominently expressed in the brain and performs an electroneutral exchange of chloride and bicarbonate ions. In order to study the potential role of AE3 in human neurological disease, we characterized AE3 genomic structure and performed mutational analysis on patients with an episodic movement disorder that maps to the same genetic locus. AE3 genomic organization, including the nucleotide sequence of the 5'-untranslated region and intron/ exon boundaries, is highly conserved between humans and homologs from mouse and rat. Mutational analysis revealed no disease-causing defect in patients with familial paroxysmal dyskinesia, although several benign polymorphisms were identified. AE3 variation may prove useful for further genetic studies, such as finer resolution mapping. Characterization of genomic structure will facilitate mutational analysis of AE3 in studies of neurological diseases mapped to the same locus.

5' Untranslated Regions↗