Identification of SNP markers for common CNV regions and association analysis of risk of subarachnoid aneurysmal hemorrhage in Japanese population

Copy number variation (CNV) is emerging as a new tool for understanding human genomic variation, but its relationship with human disease is not yet fully understood. The data for a total of 317,503 genotypes were collected for a genome-wide association study of subarachnoid aneurismal hemorrhage (SAH) in a Japanese population (cases and controls, n = 497) using Illumina HumanHap300 BeadChip^®. To identify multi-allelic CNV markers, we visually inspected all genotype clusters of 317,503 SNP markers covering the whole genome using Illumina’s BeadStudio 3.0^® software. As a result, we identified 597 multi-allelic CNV markers for common (copy loss frequency > 0.05) CNV regions in a Japanese population (n = 497). The identified CNV markers shared the following characteristics: enrichment of Hardy–Weinberg disequilibria, Mendelian inconsistency among families, and high missing genotype rate. All annotated information for those markers is summarized in our database (http://www.snp-genetics.com/user/srch.htm). In addition, we performed case-control association analyses of identified multi-allelic CNV markers with the risk of subarachnoid aneurysmal hemorrhage. One SNP marker (rs1242541) within a CNV region neighboring the Sel-1 suppressor of lin-12-like protein (SEL1L) was significantly associated with a risk of SAH (P = 0.0006). We also validated the CNV around rs1242541 using real-time quantitative polymerase chain reaction (PCR). Information and methods used in this study would be helpful for accurate genotyping of SNPs on CNV regions, which could be used for association analysis of SNP markers within CNV regions.     

Decreased rate of S-adenosyl-L-homocysteine metabolism: an early event related to transformation in cells infected with Rous sarcoma virus.

An increase of methylase activity is often related to neoplastic transformation. SAH, the natural inhibitor of transmethylases, does not inhibit cell transformation induced by RSV, in contrast to one of its synthetic analogues, SIBA. This inefficiency was thought to be due to the rapid metabolism of SAH by transformed cells. We now show, that, on the contrary, 70 % of the added amount of SAH disappears in one hour in cell-free extracts of normal cell against only 14 % in extracts of transformed cells. This decreased rate of degradation occurred one day post infection. Cells infected with the non transforming RAV₁ degrade SAH at the same rate as normal cells. A decrease of SAH-hydrolase and adenosine deaminase activity was also observed in infected cells. The decrease of the first enzyme seems to be related to the transformed state, whereas that of the second enzyme seems to depend only on infection, since it is also observed in cells infected with RAV₁.     

Identification of the Active Sites in the Methyltransferases of a Transcribing dsRNA Virus

Many double-stranded RNA (dsRNA) viruses are capable of transcribing and capping RNA within a stable icosahedral viral capsid. The turret of turreted dsRNA viruses belonging to the family Reoviridae is formed by five copies of the turret protein, which contains domains with both 7-N-methyltransferase and 2′-O-methyltransferase activities, and serves to catalyze the methylation reactions during RNA capping. Cypovirus of the family Reoviridae provides a good model system for studying the methylation reactions in dsRNA viruses. Here, we present the structure of a transcribing cypovirus to a resolution of ~ 3.8 Å by cryo-electron microscopy. The binding sites for both S-adenosyl-l-methionine and RNA in the two methyltransferases of the turret were identified. Structural analysis of the turret in complex with RNA revealed a pathway through which the RNA molecule reaches the active sites of the two methyltransferases before it is released into the cytoplasm. The pathway shows that RNA capping reactions occur in the active sites of different turret protein monomers, suggesting that RNA capping requires concerted efforts by at least three turret protein monomers. Thus, the turret structure provides novel insights into the precise mechanisms of RNA methylation.     

Homocysteine contribution to DNA damage in cystathionine β-synthase-deficient patients

High blood levels of homocysteine (Hcy) are found in patients affected by homocystinuria, a genetic disorder caused by deficiency of cystathionine β-synthase (CBS) activity, as well as in nutritional deficiencies (vitamin B₁₂ or folate) and in abnormal renal function. We previously demonstrated that lipid and protein oxidative damage is increased and the antioxidant defenses diminished in plasma of CBS-deficient patients, indicating that oxidative stress is involved in the pathophysiology of this disease. In the present work, we extended these investigations by evaluating DNA damage through the comet assay in peripheral leukocytes from CBS-deficient patients, as well as by analyzing of the in vitro effect of Hcy on DNA damage in white blood cells. We verified that DNA damage was significantly higher in the CBS-deficient patients under treatment based on a protein-restricted diet and pyridoxine, folic acid, betaine and vitamin B₁₂ supplementation, when compared to controls. Furthermore, the in vitro study showed a concentration-dependent effect of Hcy inducing DNA damage. Taken together, the present data indicate that DNA damage occurs in treated CBS-deficient patients, possibly due to high Hcy levels.     

Folate: Metabolism,genes, polymorphisms and the associated diseases

Folate being an important vitamin of B Complex group in our diet plays an important role not only in the synthesis of DNA but also in the maintenance of methylation reactions in the cells. Folate metabolism is influenced by several processes especially its dietary intake and the polymorphisms of the associated genes involved. Aberrant folate metabolism, therefore, affects both methylation as well as the DNA synthesis processes, both of which have been implicated in the development of various diseases. This paper reviews the current knowledge of the processes involved in folate metabolism and consequences of deviant folate metabolism, particular emphasis is given to the polymorphic genes which have been implicated in the development of various diseases in humans, like vascular diseases, Down's syndrome, neural tube defects, psychiatric disorders and cancers.     

Synthesis of structurally unstable type III procollagen in patients with cerebral artery aneurysm

The biosynthesis of collagen was studied in skin fibroblast cultures established from 11 patients with cerebral artery aneurysms. Six patients had familial subarachnoid hemorrhage (SAH), while five patients were considered as sporadic cases. The structural stability of the triplet-helical medium procollagen was studied by measuring the thermal denaturation temperature (T_m) of type I and type II procollagen molecules. Structural instability of type III procollagen was demonstrated in two patients with familial SAH. Te T_m of type III procollagen was 39.0°C and 39.5°C in two of the cell lines, while the control value was 40.3°C. The stability of type I procollagen did not differ from that of the controls, and the main features of the biosynthesis of collagen were similar in the aneurysm patient cell lines and in the controls. The results suggest that a structural defect of type III procollagen may serve as an etiological factor in the formation of cerebral artery aneurysms.     

The Crystal Structure of the Novobiocin Biosynthetic Enzyme NovP: The First Representative Structure for the TylF O-Methyltransferase Superfamily

NovP is an S-adenosyl-l-methionine-dependent O-methyltransferase that catalyzes the penultimate step in the biosynthesis of the aminocoumarin antibiotic novobiocin. Specifically, it methylates at 4-OH of the noviose moiety, and the resultant methoxy group is important for the potency of the mature antibiotic: previous crystallographic studies have shown that this group interacts directly with the target enzyme DNA gyrase, which is a validated drug target. We have determined the high-resolution crystal structure of NovP from Streptomyces spheroides as a binary complex with its desmethylated cosubstrate S-adenosyl-l-homocysteine. The structure displays a typical class I methyltransferase fold, in addition to motifs that are consistent with a divalent-metal-dependent mechanism. This is the first representative structure of a methyltransferase from the TylF superfamily, which includes a number of enzymes implicated in the biosynthesis of antibiotics and other therapeutics. The NovP structure reveals a number of distinctive structural features that, based on sequence conservation, are likely to be characteristic of the superfamily. These include a helical ‘lid’ region that gates access to the cosubstrate binding pocket and an active center that contains a 3-Asp putative metal binding site. A further conserved Asp likely acts as the general base that initiates the reaction by deprotonating the 4-OH group of the noviose unit. Using in silico docking, we have generated models of the enzyme-substrate complex that are consistent with the proposed mechanism. Furthermore, these models suggest that NovP is unlikely to tolerate significant modifications at the noviose moiety, but could show increasing substrate promiscuity as a function of the distance of the modification from the methylation site. These observations could inform future attempts to utilize NovP for methylating a range of glycosylated compounds.     

Aryl hydrocarbon hydroxylase and 16α-hydroxylase in cultured human lymphocytes

Aryl hydrocarbon hydroxylase and 16-hydroxylase were examined in intact, cultured human lymphocytes. The two microsomal mixed-function oxygenases had different pH optima and showed competitive inhibition for enzyme induction and activity. Population distributions were lognormal for both enzymes, giving apparent evidence for polygenic control. Induced levels of AHH were slightly higher among first-order relatives of lung or colon cancer patients than in the control group. The correlation coefficient (r) for AHH and SAH co-inducibility was –0.08, indicating no correlation and suggesting the absence of association between the two enzymes in man.Supported in part by NIH Contract NO1-CP5-5626 and NTSU Faculty Grants 34984 and 34815.National Science Foundation Predoctoral Trainee.