INSIG1 influences obesity-related hypertriglyceridemia in humans.

In our analysis of a quantitative trait locus (QTL) for plasma triglyceride (TG) levels [logarithm of odds (LOD) = 3.7] on human chromosome 7q36, we examined 29 single nucleotide polymorphisms (SNPs) across INSIG1, a biological candidate gene in the region. Insulin-induced genes (INSIGs) are feedback mediators of cholesterol and fatty acid synthesis in animals, but their role in human lipid regulation is unclear. In our cohort, the INSIG1 promoter SNP rs2721 was associated with TG levels (P = 2 x 10(-3) in 1,560 individuals of the original linkage cohort, P = 8 x 10(-4) in 920 unrelated individuals of the replication cohort, combined P = 9.9 x 10(-6)). Individuals homozygous for the T allele had 9% higher TG levels and 2-fold lower expression of INSIG1 in surgical liver biopsy samples when compared with individuals homozygous for the G allele. Also, the T allele showed additional binding of nuclear proteins from HepG2 liver cells in gel shift assays. Finally, the variant rs7566605 in INSIG2, the only homolog of INSIG1, enhances the effect of rs2721 (P = 0.00117). The variant rs2721 alone explains 5.4% of the observed linkage in our cohort, suggesting that additional, yet-undiscovered genes and sequence variants in the QTL interval also contribute to alterations in TG levels in humans.

Serotonin (5-HT) receptor 5A sequence variants affect human plasma triglyceride levels.

Neurotransmitters such as serotonin (5-hydroxytryptamine, 5-HT) work closely with leptin and insulin to fine-tune the metabolic and neuroendocrine responses to dietary intake. Losing the sensitivity to excess food intake can lead to obesity, diabetes, and a multitude of behavioral disorders. It is largely unclear how different serotonin receptor subtypes respond to and integrate metabolic signals and which genetic variations in these receptor genes lead to individual differences in susceptibility to metabolic disorders. In an obese cohort of families of Northern European descent (n = 2,209), the serotonin type 5A receptor gene, HTR5A, was identified as a prominent factor affecting plasma levels of triglycerides (TG), supported by our data from both genome-wide linkage and targeted association analyses using 28 publicly available and 12 newly discovered single nucleotide polymorphisms (SNPs), of which 3 were strongly associated with plasma TG levels (P < 0.00125). Bayesian quantitative trait nucleotide (BQTN) analysis identified a putative causal promoter SNP (rs3734967) with substantial posterior probability (P = 0.59). Functional analysis of rs3734967 by electrophoretic mobility shift assay (EMSA) showed distinct binding patterns of the two alleles of this SNP with nuclear proteins from glioma cell lines. In conclusion, sequence variants in HTR5A are strongly associated with high plasma levels of TG in a Northern European population, suggesting a novel role of the serotonin receptor system in humans. This suggests a potential brain-specific regulation of plasma TG levels, possibly by alteration of the expression of HTR5A.

Impaired preneoplastic changes and liver tumor formation in tumor necrosis factor receptor type 1 knockout mice.

Hepatic stem cells (oval cells) proliferate within the liver after exposure to a variety of hepatic carcinogens and can generate both hepatocytes and bile duct cells. Oval cell proliferation is commonly seen in the preneoplastic stages of liver carcinogenesis, often accompanied by an inflammatory response. Tumor necrosis factor (TNF), an inflammatory cytokine, is also important in liver regeneration and hepatocellular growth. The experiments reported here explore the relationship among the TNF inflammatory pathway, liver stem cell activation, and tumorigenesis. We demonstrate that TNF is upregulated during oval cell proliferation induced by a choline-deficient, ethionine-supplemented diet and that it is expressed by oval cells. In TNF receptor type 1 knockout mice, oval cell proliferation is substantially impaired and tumorigenesis is reduced. Oval cell proliferation is impaired to a lesser extent in interleukin 6 knockout mice and is unchanged in TNF receptor type 2 knockout mice. These findings demonstrate that TNF signaling participates in the proliferation of oval cells during the preneoplastic phase of liver carcinogenesis and that loss of signaling through the TNF receptor type 1 reduces the incidence of tumor formation. The TNF inflammatory pathway may be a target for therapeutic intervention during the early stages of liver carcinogenesis.

Human natural killer (NK) alloreactivity and its association with the major histocompatibility complex: ancestral haplotypes encode particular NK-defined haplotypes.

As ancestral haplotypes of the major histocompatibility complex (MHC) appear to define identical MHC haplotypes in unrelated individuals, unrelated individuals sharing the same ancestral haplotype should also share the same NK-defined allospecificities that have recently been shown to map to the human MHC. To test this prediction, multiple cell lines from unrelated individuals sharing the same ancestral haplotypes were tested for the NK-defined allospecificities. It was found that cells sharing the same ancestral haplotypes do have the same NK-defined specificities. Furthermore, the NK-defined phenotype of cells that possess two different ancestral haplotypes can be predicted from the NK-defined phenotypes of unrelated cells that are homozygous for the ancestral haplotypes concerned. Although the group 1 and 2 NK-defined allospecificities can be explained to some extent by HLA-C alleles, evidence is presented that additional genes may modify the phenotype conferred by HLA-C.

Hepatic transcription of the acute-phase alpha 1-inhibitor III gene is controlled by a novel combination of cis-acting regulatory elements.

mRNA coding for the abundant broad-range plasma proteinase inhibitor alpha 1-inhibitor III (alpha 1I3) was detected only in rat liver, while mRNA for the related proteins alpha 1-macroglobulin and alpha 2-macroglobulin was also found in a variety of nonhepatic tissues. cis-Acting control elements necessary for the hepatic transcription of alpha 1I3 were mapped by transfection and expression studies of control-region constructs in cultured hepatic and nonhepatic cells. The promoter-proximal 5'-flanking region contained four control elements, I to IV, located between -109 and -196 base pairs upstream of the transcriptional start site relevant for the hepatic transcription of this gene. Elements II and III were essential, and I and IV exerted strong modulatory effects. Elements I to III acted as positive regulators, and IV acted as a negative element. Element II contained the sequence TGGCA and is probably a binding site for a nuclear factor related to the known transcription factor NF1. The other three elements did not resemble consensus binding sites for known transcription factors that are involved in the hepatocyte-specific transcription of other well-characterized plasma protein genes, such as the prototype factor HNF-1. Thus, the alpha 1I3 gene achieves its highly hepatocyte-specific transcription through a novel combination of cis-acting control elements and trans-acting factors.

Primary Parotid Lymphoma From A Regional Cancer Center in South India.

UNLABELLED: Primary parotid lymphoma (PPL) is an unusual entity and there is limited data in Indian population. Hence we undertook this retrospective observational study of primary parotid lymphoma at our Center in Southern India. This study includes 7 consecutive cases diagnosed as PPL by tissue biopsy/superficial/deep parotidectomy confirmed by immunohistochemistry between January 2007 and December 2012. RESULTS: Median age was 54 years (range 29- 78 years), and it was more common in males. According to Ann Arbor stage, Advanced stage (stage III and IV) was seen in 2 (28.57%). According to the International Prognostic Index (IPI), most (6) were low risk (85.7%). Overall survival ranged from 1-45 months with median OS of 18 months. To conclude, PPL presents more often in early stage and low IPI score. Surgery +/- chemoimmunotherapy with radiotherapy to the parotid is the standard treatment at present.

Characterisation of the human CD30 ligand gene structure.

The gene for the human CD30 ligand was molecularly cloned, sequenced and characterised. The gene structure consisted of four exons and three intervening introns spaced over 17.1 kb of genomic DNA. The 5' flanking region of the CD30L gene was sequenced and analysis of the region revealed the presence of several regulatory regions including a poly-dT element directly upstream from the transcription start site and consensus sequences for AP4, IK2, MZF1, E47 and ELK/cETS1. The absence of a canionical TATA motif suggests CD30L gene expression is regulated by a TATA-less promoter.

Characterization of mouse glycogenin-1 cDNA and promoter region.

Glycogenin-1 is an autocatalytic, self-glucosylating protein which acts as the primer for glycogen synthesis in mammalian skeletal muscle. In this study, we have cloned the glycogenin-1 cDNA from mouse skeletal muscle. Mouse glycogenin-1 has a predicted molecular mass of 37¿ omitted¿399 Da, and the deduced amino acid sequence exhibited 87% homology with human glycogenin-1. Northern blot analysis specifically detected mouse glycogenin-1 transcript in skeletal muscle and heart, and to a lesser extent in kidney, lung and brain. 5'-RACE analysis revealed the major transcription start site to be localized 47 bp upstream of the initiation of translation codon. Sequence analysis of approximately 2 kb of the 5'-flanking region revealed potentially important regions of homology between the mouse and human glycogenin-1 promoters. Several conserved but putative elements, including a TATA box, Sp1 site, and a cyclic AMP responsive element, were observed proximal to the transcription start site. Significantly, Northern blot analysis revealed dibutyryl-cAMP treatment of cultured mouse C2C12 myotubes markedly reduced the levels of glycogenin-1 mRNA in a dose-dependent manner.

Impact of the -308 TNF promoter polymorphism on the transcriptional regulation of the TNF gene: relevance to disease.

A biallelic G (TNF1 allele) to A (TNF2 allele) polymorphism 308 nucleotides upstream from the transcription initiation site in the tumor necrosis factor (TNF) promoter is associated with elevated TNF levels and disease susceptibilities observed in human subjects. The TNF2 allele is strongly associated with the high-TNF-producing autoimmune MHC haplotype HLA-A1, B8, DR3, with elevated serum TNF levels and a more severe outcome in infectious diseases, such as cerebral malaria. A number of groups have set out to determine whether the -308 polymorphism could affect transcription factor binding and hence influence TNF transcription and expression levels. Although some studies have failed to show any functional difference between the two allelic forms, others have shown that the -308 polymorphism effected transcription factor binding to the region encompassing -308, with the region in the TNF2 allele showing altered binding characteristics. The -308 polymorphism also has been found by some groups to be functionally significant in reporter gene assays in Raji B cells, Jurkat T cells, and U937 pre-monocytic cells. Up to fivefold differences can be measured between TNF1 and TNF2 allelic constructs when the TNF 3'UTR is present, indicating a role in the expression of the polymorphism. Although controversial, the majority of the data support a direct role for the TNF2 -308 allele in the elevated TNF levels observed in TNF2 homozygotes and HLA-A1, B8, DR3 individuals. Elevated TNF levels due to the -308 polymorphism may alter the immune response such that it confers susceptibility to certain autoimmune and infectious diseases.

The -308 tumor necrosis factor-alpha promoter polymorphism effects transcription.

Since the tumor necrosis factor alpha (TNF-alpha) gene was found to be located in the central major histocompatibility complex (MHC) there has been much speculation concerning a genetic association between particular TNF alleles and disease susceptibility. A relationship between the MHC haplotype A1, B8, DR3, TNF-alpha expression levels and susceptibility to autoimmune disease has been suggested by several groups. The identification of the -308 polymorphism and its association with the HLA A1, B8, DR3 haplotype have led to speculation that the polymorphism may play a role in the altered expression of TNF-alpha. We have demonstrated that the region (-323 to -285) encompassing -308 in the TNF2 allele binds nuclear factors differently to the same region in the promoter of the more common TNF1 allele. The G/A -308 polymorphism affected the affinity of factor binding and resulted in a factor binding to TNF2 but not TNF1. The observed differential binding was shown to be functional, with the 38bp region from TNF2 causing a two-fold greater activity of a heterologous promoter over that due to the same region in TNF1. To further substantiate the functional consequences of the TNF-alpha -308 polymorphism, we analysed both allelic forms of the TNF-alpha promoter region (-993 to +110) in a transient transfection assay, using luciferase as a reporter gene. The results showed that when present with the 3'UTR the -308A allelic form gave a two-fold greater level of transcription than the 308G form in PMA-stimulated Jurkat and U937 cells. This suggests that the -308 G/A polymorphism may play a role in the altered TNF-alpha gene expression observed in individuals with the HLA A1, B8, DR3 haplotype.

Characterization of the ush gene of Escherichia coli and its protein products.

The Escherichia coli ush gene has been subcloned and the coding sequence delineated using BAL31 nuclease digestion. Synthesis of proteins encoded by the ush gene have been examined in "maxicells"; two proteins are made, one of which corresponds in Mr (61000) to purified uridine diphosphoglucose hydrolase and the other, less abundant, has an Mr of 43 000. A deletion at the 3' end of the gene introduced by restriction endonuclease digestion, results in the synthesis of a truncated protein of the expected Mr of about 43 000. Precursors of all these proteins are observed in maxicells under conditions known to inhibit processing of secreted proteins. Whereas the precursor of the major ush-encoded protein is retained in the cytoplasm-plus-membrane fraction, unexpectedly the precursor of the truncated protein is secreted. The mature forms of both the normal and truncated proteins are secreted.

Characterization of the human glycogenin-1 gene: identification of a muscle-specific regulatory domain.

The de-novo synthesis of glycogen is now known to involve a novel class of self-glucosylating protein primers. In mammalian skeletal muscle, glycogenin-1 is the protein responsible for this initiation step. Northern blot analysis revealed that glycogenin-1 gene transcription is differentially regulated in the C2C12 mouse muscle cell line. To define the regulatory elements that control expression of the glycogenin-1 gene, we have cloned and characterized the genomic structure of the human glycogenin-1 gene and its promoter region. This gene consists of seven exons and six introns, and spans over 13kb. Transcription of human glycogenin-1 is initiated at two major sites, 80 and 86bp upstream from the initiation of translation codon. Nucleotide sequence analysis of 2.1kb of the 5'-flanking region revealed the proximal promoter contains both a TATA box and two putative Sp1 binding sites located in a CpG island. There are numerous binding sites for developmental and cell-type-specific transcription factors, including AP-1, AP-2, GATA, and several potential Oct 1 binding domains. There are also nine consensus E-boxes that bind the basic helix-loop-helix family of muscle-specific transcription factors. The transcriptional activity of the glycogenin-1 gene was investigated by transient transfection of the 5'-flanking region in HepG2 cells and C2C12 myoblasts and myotubes. These results permitted the definition of a minimal 232bp promoter fragment that is responsible for basal level transcription in a cell-type-independent manner. Furthermore, we have identified a regulatory region located between -2076 and -1736 of the 5'-flanking region of the human glycogenin-1 gene that allows myotube-specific expression in C2C12 cells.

Analysis of the human and mouse promoter region of the non-Hodgkin's lymphoma-associated CD30 gene.

To investigate the regulation of CD30 at the level of transcription, we have isolated and compared the promoter sequence of human and murine CD30. Analysis of the human and mouse promoter identified a number of potential transcription factor binding sites, including ETS, MZF, AP-1, IK2, CREB, Stat, USF, and Spl. The absence of TATA or CAAT boxes and the identification of one major and three minor transcription initiation sites for CD30 suggest that it is a member of the class of TATA-less promoters that use initiator elements to correctly position the RNA polymerase. Comparison of the murine and human CD30 promoters identified a number of highly conserved regions, including an Spl site 40 bp upstream from the major start site and a downstream promoter element (DPE) that may be involved in directing transcriptional initiation of the CD30 gene. Functional analysis of the human CD30 promoter in transfected Jurkat T cells provided further evidence that these conserved regions are important regulatory elements in the CD30 promoter.

Regulation of lymphotoxin-beta by tumor necrosis factor, phorbol myristate acetate, and ionomycin in Jurkat T cells.

Lymphotoxin-beta (LT- beta) is a tumor necrosis factor (TNF)-related membrane-bound cytokine that forms a heterotrimeric surface lymphotoxin (LT) complex with LT-alpha on the surface of lymphoid cells. Although knockout studies have revealed a role in lymph node biogenesis during development, the regulation and function of surface LT in mature cell types are poorly understood. The present study aims to understand the physiologic signals that regulate the components of surface LT in Jurkat T cells. We show that the previously observed upregulation of surface LT by phorbol myristate acetate (PMA) is markedly abrogated by cotreatment with ionomycin through posttranscriptional mechanisms. In addition, the observation of striking similarities between the mRNA accumulation kinetics of LT-alpha and LT-beta during these treatments indicates tight coupling of expression under certain conditions. In investigating the reported upregulation of LT-beta during inflammation, we tested the effects of various proinflammatory and anti-inflammatory cytokines on LT-beta expression. Our data demonstrate an upregulation of LT-beta mRNA by the inflammatory cytokines TNF and LT-alpha.

New major histocompatibility complex genes.

The MHC is a region of some 4 megabases that has been studied intensively owing to the large number of diseases that are associated with susceptibility genes within this region of the genome. The total number of genes located within the MHC is now approximately 100, but more can be predicted. Recently identified genes within the MHC include PERB6, a large gene producing multiple transcripts located between HLA-B and TNF, and PERB1, a member of the protein tyrosine kinase-gene family. PERB6 was identified by YAC probing of tissue blots, while PERB1 was identified by genomic sequencing.

Differences in the central major histocompatibility complex between humans and chimpanzees. Implications for development of autoimmunity and acquired immune deficiency syndrome.

Chimpanzees (Pan Troglodytes) and humans are closely related and belong to the same subfamily, Homininae. The approximately 1.8% genetic difference that exists between humans and the chimpanzees must be responsible for observed differences between these two species. It has been shown that chimpanzees can be infected with HIV, but AIDS has not been reported. Furthermore, the prevalence of autoimmune diseases may be low in this species. For instance, type II diabetes occurs, but type I (autoimmune) diabetes (IDDM), to our knowledge, has not been reported. In humans, susceptibility genes for MG and IDDM have been localized to the region between TNF and HLA-B. This region may also influence the rate of progression to death after HIV infection. We have identified differences in this region between humans and the chimpanzees. As shown by PFGE, the TNF to Patr-B region in the chimpanzees is approximately 130-160 kb shorter than the equivalent in humans. Southern and sequence analyses indicate that the deletions in chimpanzees (insertions in humans) include one copy of CL (approximately 10 kb) and the X sequences (< 30 kb). Obviously, other deletions/insertions (approximately 120 kb) need to be identified. Since CL has been shown to be transcribed, the results imply the lack of the gene or, at least, a different gene copy number in the chimpanzees, and we propose that such differences may be relevant to the observed functional differences. We demonstrate here a strategy to identify critical genes responsible for disease development.

Haplospecific polymorphism between HLA B and tumor necrosis factor.

Polymorphisms were sought between HLA B and tumor necrosis factor (TNF) using three genomic probes. Extensive polymorphism was detected within a panel of 50 cell lines including 37 homozygotes representing 21 different ancestral haplotypes (AH). Following Taq I digestion of genomic DNA, we observed three allelic patterns with probe X (R17A) and four with probe V (R9A). Seven different allelic patterns were found with probe Y (M20A) after Taq I + Rsa I digestion. Family studies showed that the Y, X, and V alleles were inherited and segregated with HLA haplotypes. A striking feature of the allelic patterns detected by these probes was that cells with the same AH had identical Y, X, and V alleles (i.e., the alleles were haplotypic). Of 15 different Y-X-V haplotypes observed, 11 were found to be specific for a particular AH (i.e., were haplospecific). Four were shared by more than one AH, but in these instances there were extensive similarities in other regions within the major histocompatibility complex (MHC), for example, the Japanese 46.2 (HLA Bw46-DRw8) and the Chinese 46.1 (Bw46-DR9) share all alleles between HLA C and C4 and differ only in class II, suggesting their relatively recent divergence by recombination between C4 and DR. Surprisingly, two insulin-dependent diabetes mellitus (IDDM)-resistant but race-specific AHs 52.1 (Bw52-DRB1*1502, Japanese) and 7.1 (B7-DRB1*1501, Caucasoid) carry the same Y-X-V haplotype, suggesting the possibility of localizing gene(s) relevant to IDDM. The present study confirms that MHC AHs have been conserved en bloc, including the region between HLA B and TNF.

Ancestral haplotypes: conserved population MHC haplotypes.

We describe here a number of Caucasoid MHC haplotypes that extend from HLA-B to DR and that have been conserved en bloc. These haplotypes and recombinants between any two of them account for 73% of unselected haplotypes in our Caucasoid population. The existence of ancestral haplotypes implies conservation of large chromosomal segments. Irrespective of the mechanisms involved in preservation of ancestral haplotypes, it is clear that these haplotypes carry several MHC genes, other than HLA, which may be relevant to antigen presentation, autoimmune responses, and transplantation rejection. In light of the existence of ancestral haplotypes, it is critical to evaluate MHC associations with disease and transplantation outcome in terms of associations with ancestral haplotypes rather than individual alleles.

A region centromeric of the major histocompatibility complex class I region is as highly polymorphic as HLA-B. Implications for recombination.

Two hallmarks of the MHC are the high degree of polymorphism apparent at multiple loci and "linkage disequilibrium." The data presented here suggest that a consequence of selection at a particular locus may be the inhibition of recombination through the accumulation of DNA sequence polymorphisms. Equivalent 6.4 kb regions from a locus, CL1, located approximately 25-30 kb centromeric of HLA-B, were sequenced for three ancestral haplotypes: A1,B8,DR3; A30,B18,DR3; and A1,B57,DR7. Comparison of the sequences indicated that the level of DNA sequence polymorphism was high when compared with the TNF region; approximately 80 single nucleotide differences were found when comparing any two sequences. In addition, multiple deletions/insertions were present. We believe that the degree of polymorphism within the CL interval may be adequate to at least partially inhibit recombination between the haplotypes studied.

Microsatellite, restriction fragment-length polymorphism, and sequence-specific oligonucleotide typing of the tumor necrosis factor region. Comparisons of the 4AOHW cell panel.

The location of the TNF and other genes in the central MHC and their possible relevance to disease susceptibility provided an impetus to develop useful typing markers. The 4AOHW undertook to assess the various markers available, including DNA sequence-based systems. A panel of well-characterized lymphoblastoid cell lines were typed by Nco I RLFP analysis, SSO typing, and TNF microsatellite typing. RFLP and SSO typing were relatively reproducible as judged by the blind replicates. The two techniques provided the same results with only one exception, and it would be reasonable to prefer SSO typing because of its advantages in terms of cost and time. Microsatellite typing was much more discriminating but, as expected, less robust in that some discrepancies were apparent. As a result of the workshop and subsequent testing, alleles and haplotypes were allocated to most cells within the 4AOHW panel, including 10W cells typed in previous studies. While there was evidence that microsatellites may be relatively stable, they have the potential to identify recent mutations within ancestral haplotypes.