Estimating haplotype frequencies and standard errors for multiple single nucleotide polymorphisms

Estimating haplotype frequencies becomes increasingly important in the mapping of complex disease genes, as millions of single nucleotide polymorphisms (SNPs) are being identified and genotyped. When genotypes at multiple SNP loci are gathered from unrelated individuals, haplotype frequencies can be accurately estimated using expectation-maximization (EM) algorithms (Excoffier and Slatkin, 1995; Hawley and Kidd, 1995; Long et al., 1995), with standard errors estimated using bootstraps. However, because the number of possible haplotypes increases exponentially with the number of SNPs, handling data with a large number of SNPs poses a computational challenge for the EM methods and for other haplotype inference methods. To solve this problem, Niu and colleagues, in their Bayesian haplotype inference paper (Niu et al., 2002), introduced a computational algorithm called progressive ligation (PL). But their Bayesian method has a limitation on the number of subjects (no more than 100 subjects in the current implementation of the method). In this paper, we propose a new method in which we use the same likelihood formulation as in Excoffier and Slatkin's EM algorithm and apply the estimating equation idea and the PL computational algorithm with some modifications. Our proposed method can handle data sets with large number of SNPs as well as large numbers of subjects. Simultaneously, our method estimates standard errors efficiently, using the sandwich-estimate from the estimating equation, rather than the bootstrap method. Additionally, our method admits missing data and produces valid estimates of parameters and their standard errors under the assumption that the missing genotypes are missing at random in the sense defined by Rubin (1976).     

Anti-P53 antibodies in Brazilian brain tumor patients

Gliomas of astrocytic origin are the most common primary brain tumors, accounting for over 40 to 50% of all central nervous system tumors. The TP53 tumor suppressor gene is the most frequently mutated gene found in human malignancies. A mutation of this gene can lead to an increased half-life of the resulting protein and loss of biological function. High levels of p53 have been detected in the serum of colon cancer patients, although p53 protein has not been detected in the serum of brain tumor patients. Besides circulating p53, several studies have detected antibodies against p53 in patients with lung and breast cancer, as well as those with other types of cancer. We studied p53 protein and anti-p53 antibodies in the plasma of Brazilian brain tumor patients. Plasma samples were drawn from 24 untreated brain tumor patients and from 15 healthy donors without clinical signs of cancer. Western blotting techniques were used to detect p53 protein and anti-p53 antibodies. We found anti-p53 antibodies in 5/24 brain tumor patients. Age appears to affect the immune response, as four of six tumor patients under 16 years old had detectable anti-p53 antibodies, while these were found in only 1 of 18 adults (over 16 years old). We found no p53 protein in any of the serum samples from the brain tumors. Possibly the presence of this protein is affected by tumor type or by the organs that are sampled.     

p53 polymorphisms in Russia and Belarus: correlation of the 2-1-1 haplotype frequency with longitude

Four different polymorphisms in the human p53 gene (a 16-bp duplication in intron 3, and three RFLPs: for Bsh1236I at codon 72, for MspI in intron 6 and for BamHI in the 3 flanking region) and extended haplotypes were studied in nine geographically diverse populations from Russia and Belarus. The Yakuts differed from all other populations, as they had a significantly higher frequency of the BamHI A1 allele. Most populations did not differ significantly from each other in the frequency of the Bsh1236I polymorphism. The 16-bp duplication A1 allele and MspI A2 allele frequencies were significantly higher in the Yakut and Khant populations. Linkage disequilibrium values (D) between BamHI and other polymorphic sites were not significant in many cases; for this reason we have used the 16 bp–Bsh1236I–MspI haplotype frequencies only. Of eight possible haplotypes, five were observed in the populations investigated. Haplotype 1-2-2 was the most frequent in all populations. The next most common haplotype, 1-1-2, was present at very similar frequencies among the Byelorussians and Russians from Smolensk, but was more frequent in other populations. The frequency of haplotype 2-1-1 showed a nearly continuous decrease from West to East (from 17.857% among the Byelorussians to 0.685% in the Yakuts from the Verkhoyansk) and correlated with longitude (Spearmans r=–0.8667, P=0.0025), which may be due to natural selection and adaptation. The relationships among populations were evaluated by means of Neis D_A distances for the 16 bp–Bsh1236I–MspI haplotype frequencies. Based on the multidimensional scaling analysis a correlation between p53 haplotype frequencies and ethnicity is supposed.     

Drosophila p53 is a structural and functional homolog of the tumor suppressor p53

The importance of p53 in carcinogenesis stems from its central role in inducing cell cycle arrest or apoptosis in response to cellular stresses. We have identified a Drosophila homolog of p53 ("Dmp53"). Like mammalian p53, Dmp53 binds specifically to human p53 binding sites, and overexpression of Dmp53 induces apoptosis. Importantly, inhibition of Dmp53 function renders cells resistant to X ray-induced apoptosis, suggesting that Dmp53 is required for the apoptotic response to DNA damage. Unlike mammalian p53, Dmp53 appears unable to induce a G1 cell cycle block when overexpressed, and inhibition of Dmp53 activity does not affect X ray-induced cell cycle arrest. These data reveal an ancestral proapoptotic function for p53 and identify Drosophila as an ideal model system for elucidating the p53 apoptotic pathway(s) induced by DNA damage.     

Allelic frequencies of FBN1 gene polymorphisms and genetic analysis of italian families with Marfan syndrome

The fibrillin gene (FBN1) is the disease locus for Marfan syndrome. This disorder shows a high degree of clinical and allelic heterogeneity. Direct mutation screening has proven difficult and inefficient and at present cannot be utilized for routine analysis. In familial cases linkage analysis represents a useful tool for molecular diagnosis. We have determined the allelic frequencies of 5 polymorphic markers within the FBN1 locus in the Italian population and have successfully employed them for prenatal diagnosis and resolution of clinically equivocal cases.     

Allelic expression of the putative tumor suppressor gene p73 in human fetal tissues and tumor specimens

p73, a proposed tumor suppressor, shares significant amino acid sequence homology with p53. However, p73 is rarely mutated in tumors but it has been suggested that p73 is monoallelically expressed in some tissues. This latter feature would predispose p73 to gene inactivation because a single genetic 'hit' or the loss of the expressed parental allele would leave the cell without p73 activity. We examined the allelic expression of p73 in normal fetal tissues and in ovarian cancer and Wilms' tumor. We found that p73 was biallelically expressed in all fetal tissues, except in brain, where differential expression of the two parental alleles was observed. Biallelic expression of p73 was also observed in paired samples of ovary cancer and Wilms' tumor. Loss of heterozygosity of p73 occurred at relatively low rates in tumors: one of 11 informative samples (9.1%) of ovarian cancer and two of 19 (10.1%) Wilms' tumors. These data demonstrate that p73 is biallelically expressed in most tissues, thus excluding genomic imprinting as a molecular mechanism to predispose to allelic inactivation of p73 in human tumors.     

Glutathione S-transferase GSTM1, GSTT1 and p53 codon 72 polymorphisms in human tumor cells

The genes of the glutathione S-transferase (GST) family encode enzymes that appear to be critical in cellular protection against the cytotoxic effects, whereas p53 is a tumor suppressor gene. Despite a large number of studies on germline polymorphisms of GSTM1, GSTT1 and p53 genes, there have been very few reports on genotyping of these genes in human malignant tumor cells. In this study, we investigated GSTM1, GSTT1 and p53 codon 72 polymorphisms in a variety of human tumor cell lines originating from different organs to clarify tissue-specific polymorphic frequency of these genes in human solid tumors. The GSTM1 and GSTT1 genetic polymorphisms were evaluated using multiplex PCR techniques and PCR-RFLP analysis was conducted to identify p53 codon 72 genotypes. Gene expression of GSTM1 or GSTT1 was detected by RT-PCR in the cells with respective present genotype for each. Polymorphisms of p53 codon 72 detected by PCR-RFLP were also confirmed using SSCP and sequence analyses. GSTM1 and GSTT1 genotypes were various in 104 cell lines examined. Null GSTM1 genotype was dominant in small cell lung, kidney and ovarian carcinoma cells, whereas null GSTT1 genotype was dominant in cervical and endometrial carcinoma cells. GSTM1 and GSTT1 genotypes in ovarian carcinoma cells were quite similar to those in small cell lung carcinoma cells. Polymorphic frequency of p53 codon 72 was also various among the cells, however, the Pro allele was found in only 1 of 6 kidney, 14 cervical and 4 endometrial carcinoma cell lines. There was a significant difference in GSTM1 and p53 genotypes between 34 small cell and 24 non small cell lung carcinoma cells (P < 0.01). Combined study on the distribution of GSTM1, GSTT1 and p53 genotypes revealed that null GSTM1 genotype was associated with the Arg allele of p53 codon 72 in 58 lung carcinoma cells and null GSTT1 genotype was associated with the Pro/Pro homozygote in 104 tumor cell lines examined. This is the first study examining GSTM1, GSTT1 and p53 codon 72 polymorphisms in a variety of human solid tumor cells and suggesting that polymorphic frequency of these genes may be tissue- and organ-specific. The molecular interaction between GST gene defects and p53 codon 72 genotype in the development of human malignant tumors should be further investigated.     

HLA gene and haplotype frequencies in Uruguay

A total of 604 individuals living in Montevideo and other places in Uruguay were studied in relation to three I HLA loci. The most common alleles observed (percentages in parentheses) were A2(24), A9(15), A19(11), B12(12), B35(12), and C4(16). The most marked departures from linkage equilibrium (all numbers multiplied by 10⁵) were B35-C4(636), A2-B5(590), A2C3(515), A2B14(494), and A19-B12(485). These findings do not contradict the hypothesis that while most of the Uruguayan population is of Caucasoid origin, significant African and Amerindian genes may exist in its gene pool.     

p53, Autophagy and tumor suppression

Autophagy was recently established as a novel tumor suppression mechanism, which stimulated a wave of investigations that were aimed at understanding exactly how autophagy prevents tumorigenesis, as well as to determine to what extent autophagy is implicated in human cancers. Autophagy might exert its tumor suppression function at the subcellular level by removing defective cytoplasmic components, such as damaged mitochondria. In addition, it might function at the cellular level by helping in the orderly removal of damaged cells. Previous studies indicated that autophagy is compromised in human breast, ovarian and prostate cancers. Recent research revealed that autophagy is activated by p53, a critical tumor suppressor that is involved in most, if not all, tumorigenesis. This study places autophagy in a broader context of human cancers. Future work elucidating the role of autophagy in the p53 circuit and p53 function might provide more insight into tumorigenesis and targeted cancer chemotherapy.     

Activation of the p53 tumor suppressor protein

The p53 tumor suppressor gene plays an important role in preventing cancer development, by arresting or killing potential tumor cells. Mutations within the p53 gene, leading to the loss of p53 activity, are found in about half of all human cancers, while many of the tumors that retain wild type p53 carry mutations in the pathways that allow full activation of p53. In either case, the result is a defect in the ability to induce a p53 response in cells undergoing oncogenic stress. Significant advances have been made recently in our understanding of the molecular pathways through which p53 activity is regulated, bringing with them fresh possibilities for the design of cancer therapies based on reactivation of the p53 response.     

DNA damage,p53 gene expression and p53 protein level in the rat brain aging

The aging induces free radicals leading to DNA damage (8‐oxo‐2′‐deoxyguanosine, 8‐oxo2dG). DNA injury causes increased expression of p53 gene and p53 protein. Levels of 8‐oxo2dG (HPLC), p53 mRNA (PCR) and p53 protein (Western blot) were estimated in gray matter (GM), white matter (WM), cerebellum (C) and medulla oblongata (MO) of control, 12‐ and 24‐month‐old rats. The level of 8‐oxo2dG increased with age in C (P < 0.05 in 12‐month‐old and P < 0.01 in 24‐month‐old rats) and MO. In 12‐month‐old animals the level of 8‐oxo2dG in GM and WM was higher than in controls. In 12‐month‐old animals p53 gene expression decreased while amounts of p53 protein increased, depending on the oxidative DNA damage. In 24‐month‐old rats, expression of p53 increased in all structures (P ≤ 0.05) while p53 protein showed decreased levels in most of structures of central nervous system (WM, C, MO). Aging leads to increased 8‐oxo2dG and augmented p53 gene expression, accompanied by a lowered expression of p53 protein.     

The p53 tumor suppressor gene

p53 was originally considered to be a nuclear oncogene, but several convergent lines of research have indicated that the wild-type gene functions as a tumor suppressor gene negatively regulating the cell cycle. Mutations in the p53 gene have been detected in many tumor types and seem to be the most common genetic alterations in human cancer. In this preliminary study, sera of 92 patients (pts) with breast disease were analyzed for the presence of the mutant p53 protein (mp53) with a selective immunoenzyme assay employing a monoclonal antibody (PAb 240) specific for the majority of mammalian m p53 but not for the wild-type protein. Of the 10 patients with benign breast disease, only two (20%) showed detectable m p53 levels in the serum. In the breast cancer group, sera from 7 of the 30 pts (23%) without lymph node involvement were positive for m p53, as were 7 out of the 45 pts (15%) with metastatic lymph nodes and 1 out of the 7 pts (14%) with disseminated disease. The specifity of m p53 assay evaluated in 20 healthy controls was 100%. These preliminary results showed that serum positivity for m p53 is not related to breast disease extension. Further studies to assess the utility of m p53 as a possible prognosis factor in breast cancer are currently in progress.     

Estimation of HLA phenotype frequencies from two- and three-locus haplotype frequencies

A procedure for estimating HLA phenotype frequencies from two- and three-locus haplotype frequencies is described. Formulae for this procedure are derived, and examples are presented to illustrate the application. The procedure is useful when multiple-locus phenotype frequencies from a laboratory control series are not available, or when a sufficiently large number of laboratory controls have not yet been typed for recently defined antigens or loci to yield stable multiple-locus rates for comparative purposes.     

Pituitary tumor transforming gene causes aneuploidy and p53-dependent and p53-independent apoptosis

The pituitary tumor transforming gene, PTTG, is abundantly expressed in several neoplasms. We recently showed that PTTG overexpression is associated with apoptosis and therefore have now studied the role of p53 in this process. In MCF-7 breast cancer cells that express wild type p53, PTTG overexpression caused apoptosis. p53 was translocated to the nuclei in cells expressing PTTG. Overexpression of p53, along with PTTG, augmented apoptosis, whereas expression of the human papillomavirus E6 protein inhibited PTTG-induced apoptosis. In MG-63 osteosarcoma cells that are deficient in p53, PTTG caused cell cycle arrest and subsequent apoptosis that was inhibited by caspase inhibitors. A proteasome inhibitor augmented PTTG expression in stable PTTG transfectants, suggesting that down-regulated PTTG expression is required for cell survival. Finally, MG-63 cells expressing PTTG showed signs of aneuploidy including the presence of micronuclei and multiple nuclei. These results indicate that PTTG overexpression causes p53-dependent and p53-independent apoptosis. In the absence of p53, PTTG causes aneuploidy. These results may provide a mechanism for PTTG-induced tumorigenesis whereby PTTG mediates aneuploidy and subsequent cell transformation.