Two genes encoding immune-regulatory molecules (LAG3 and IL7R) confer susceptibility to multiple sclerosis.

Multiple sclerosis (MS) is a T-cell-mediated disease of the central nervous system, characterized by damage to myelin and axons, resulting in progressive neurological disability. Genes may influence susceptibility to MS, but results of association studies are inconsistent, aside from the identification of HLA class II haplotypes. Whole-genome linkage screens in MS have both confirmed the importance of the HLA region and uncovered non-HLA loci that may harbor susceptibility genes. In this two-stage analysis, we determined genotypes, in up to 672 MS patients and 672 controls, for 123 single-nucleotide polymorphisms (SNPs) in 66 genes. Genes were chosen based on their chromosomal positions or biological functions. In stage one, 22 genes contained at least one SNP for which the carriage rate for one allele differed significantly (P<0.08) between patients and controls. After additional genotyping in stage two, two genes--each containing at least three significantly (P<0.05) associated SNPs--conferred susceptibility to MS: LAG3 on chromosome 12p13, and IL7R on 5p13. LAG3 inhibits activated T cells, while IL7R is necessary for the maturation of T and B cells. These results imply that germline allelic variation in genes involved in immune homeostasis--and, by extension, derangement of immune homeostasis--influence the risk of MS.

APOE genotypes and disease severity in multiple sclerosis.

Apolipoprotein E (opoE) is involved in the transport of lipids necessary for membrane repair and is encoded by a gene on chromosome 19q13, a region positive for linkage in two multiple sclerosis (MS) genome-wide screens. The APOE epsilon4 allele confers susceptibility to both familial and sporadic Alzheimer's disease (AD). Carriage of epsilon4 is associated with defective dendritic remodeling in AD, and with unfavorable clinical outcome in head trauma and cerebrovascular disease. According to the results of previous studies, APOE epsilon4 does not increase the risk of developing MS, but it may influence disease progression and ultimate disability. From a total cohort of over 900 MS patients, we compared APOE epsilon2-4 genotypes in, roughly, the cohort's least disabled and most disabled septiles. 'Benign MS' (n=124) was defined as an Expanded Disability Status Scale (EDSS) score of 3.0 or less, despite at least 10 years of disease duration, and 'severe MS' (n=140) as the attainment of an EDSS score of 6.0 within 8 years of disease onset. We found no significant differences in genotype or phenotype frequencies between the benign-MS and severe-MS septiles; however, the risk conferred by epsilon4 rose progressively upon comparison of carriage rates in more narrowly defined anti-podal quantiles.

Does 77C-->G in PTPRC modify autoimmune disorders linked to the major histocompatibility locus?

A 77G allele of the gene encoding CD45, also known as the protein tyrosine phosphatase receptor-type C gene (PTPRC), has been associated with multiple sclerosis (MS). Here we determine allele frequencies in large numbers of MS patients, primary immunodeficiencies linked to major histocompatibility complex (MHC) locus and over 1,000 controls to assess whether aberrant splicing of PTPRC caused by the 77C-->G polymorphism results in increased susceptibility to these diseases. Our results show no difference in the frequency of the 77G allele in patients and controls and thus do not support a causative role for the polymorphism in the development of disorders with a strong autoimmune component in etiology.

Effects of HsRad51 overexpression on cell proliferation, cell cycle progression, and apoptosis.

Expression of the DNA repair and recombination protein human Rad51 (HsRad51) is increased in transformed cells and in cancer cell lines. In order to study the effects of acute HsRad51 ectopic overexpression on cell proliferation, cell cycle progression, and apoptosis, we generated clones of the human fibrosarcoma cell line HT1080 carrying a HsRad51 transgene under a repressible promoter. The HsRad51-overexpressing cells showed decreased plating efficiency and growth rate in a dose-dependent manner with regard to the degree of overexpression. An accumulation of HsRad51-overexpressing cells in G(2) was observed following release of cells after synchronization with double thymidine block. Moreover, the fraction of apoptotic cells measured by annexin V-FACS increased with the time of HsRad51 overexpression. In the light of these observations, sustained increased levels of HsRad51 may contribute to tumor progression by causing a selection for cells tolerant to the growth-suppressive and apoptosis-inducing effects of acute HsRad51 overexpression.

Caspase-3 mediated cleavage of HsRad51 at an unconventional site.

The human recombinase HsRad51 is cleaved during apoptosis. We have earlier observed cleavage of the 41-kDa full-length protein into a 33-kDa product in apoptotic Jurkat cells and in in vitro translated HsRad51 after treatment with activated S-100 extract. In this study, site-directed mutagenesis was used for mapping of the cleavage site to AQVD274 downward arrow G, which does not correspond to a conventional caspase cleavage site. The absence of HsRad51 cleavage in staurosporine-treated apoptotic MCF-7 cells, which lack caspase-3, indicates that caspase-3 is essential for HsRad51 cleavage in vivo. Cleavage into the 33-kDa fragment was generated by recombinant caspase-3 and -7 in in vitro translated wild type HsRad51, but not in the HsRad51 AQVE274 downward arrow G mutant. Similarly, HsRad51 of Jurkat cell extracts was cleaved into the 33-kDa product by recombinant caspase-3, whereas caspase-7 failed to cleave endogenous HsRad51. The cleavage of in vitro translated wild type and AQVE274 downward arrow G mutant HsRad51 as well as of endogenous HsRad51 also gave rise to a smaller fragment, which corresponds in size to a recently reported DVLD187 downward arrow N HsRad51 cleavage product. In Jurkat cell extracts, the AQVD274 downward arrow G and DVLD187 downward arrow N cleavage products of HsRad51 appeared at equal concentrations of caspase-3. Moreover both fragments were generated by induction of apoptosis in MDA-MB 157 cells with staurosporine and in Jurkat cells with camptothecin. Thus, two sites in the HsRad51 sequence are targets for caspase cleavage both in vitro and in vivo.

p53 splice acceptor site mutation and increased HsRAD51 protein expression in Bloom's syndrome GM1492 fibroblasts.

GM1492 human diploid skin fibroblasts derived from a patient with Bloom's syndrome (BS), lack detectable p53 mRNA and protein as shown by Northern and Western blotting, and express an increased RecA-like activity. Here we demonstrate that the p53 gene is grossly intact in GM1492 cells according to Southern blotting. DNA sequencing did not reveal any mutations in the promoter region of p53. A highly sensitive RT-PCR produced a p53 cDNA fragment that was shorter than expected. DNA sequence analysis of p53 cDNA showed that exon 6 was missing, explaining the shorter PCR product. Furthermore, sequencing of genomic DNA revealed a base substitution at the nucleotide preceding the AG splice acceptor site of intron 5. The omission of exon 6 creates a frameshift at the junction of exons 5 and 7, and a premature stop codon in exon 7. The aberrant transcript is predicted to encode a truncated p53 protein containing 189 amino acid residues. Moreover, Western blotting demonstrated elevated HsRAD51 protein levels in GM1492 cells. The lack of sufficient levels of wild-type p53 and increased levels of HsRad51 protein may contribute to the elevated RecA-like activity in the GM1492 fibroblasts.

Missense mutations in hMLH1 associated with colorectal cancer.

One of the most prevalent hereditary syndromes associated with colorectal cancer is hereditary nonpolyposis colorectal cancer (HNPCC). The inherited gene defects in HNPCC have been shown to reside in DNA mismatch repair genes, mostly hMSH2 or hMLH1. Most HNPCC patients are heterozygous with regard to the relevant mismatch repair gene; they have one normal and one mutated allele, and mismatch repair in normal somatic cells is functional. Cancer predisposition in HNPCC is believed to be associated with the loss of the wild-type allele in somatic cells, resulting in defective DNA mismatch repair. This gives rise to DNA microsatellite instability (MSI), an increased somatic mutation rate, and eventually, to the accumulation of mutations in genes involved in colorectal carcinogenesis. In support of this theory, colorectal tumors in HNPCC patients and in mice deficient for hMSH2 or hMLH1 show MSI. Here, we describe two missense mutations in hMLH1 exon 16 associated with colorectal cancer. Interestingly, the tumors do not show MSI. This raises some potentially important issues. First, even microsatellite-negative colorectal tumors can be associated with germline mutations and these will be missed if an MSI test is used to select patients for mutation screening. Second, the lack of MSI in these cases suggests that the mechanism involved in carcinogenesis could be different from that generally hypothesized.

The frequency of illegitimate TCRbeta/gamma gene recombination in human lymphocytes: influence of age, environmental exposure and cytostatic treatment, and correlation with frequencies of t(14;18) and hprt mutation.

Chromosome translocations in lymphoid malignancies often involve V(D)J recombinase mediated events giving rise to aberrant T-cell receptor (TCR) and immunoglobulin genes, which have been suggested to be useful as markers of genomic instability, genotoxic exposure and cancer risk. Illegitimate rearrangements involving the TCRbeta/gamma loci on chromosome 7 create TCRbeta/gamma hybrid genes which occur at low frequency in peripheral blood lymphocytes (PBLs) of normal healthy individuals. To evaluate the utility of this marker, we studied the possible effects of age and genotoxic exposures on the TCRbeta/gamma gene variant frequency (VF), and compared the frequencies of hypoxanthine guanine phosphoribosyl transferase (hprt) mutation, hprt exon 2/3 deletion, t(14;18) and TCRbeta/gamma gene rearrangements in cells from the same donors. The TCRbeta/gamma VF ranged five-fold among 16 middle aged blood donors with a mean of 0.74+/-0.29/10(5) PBLs, which is consistent with our previous estimate in healthy subjects. The TCRbeta/gamma VF was found to increase from birth until early adult life, and then to decrease with increasing age. Four testis cancer patients, who 6 years earlier had been treated with etoposide and other cytostatic drugs, showed TCRbeta/gamma VF similar to that in healthy controls. No increase of the TCRbeta/gamma VF was found among non-smoking PAH-exposed aluminum smelter workers compared to non-smoking controls. Smoking smelter workers showed decreased TCRbeta/gamma VF compared to non-smoking workers and controls, but in a follow-up study 2 years later the difference was no longer statistically significant, although the smoking smelter workers still showed a lower TCRbeta/gamma VF than the controls. No correlation was obtained between the TCRbeta/gamma VF and the t(14;18) or hprt mutant frequency (MF) in a group of healthy individuals. However, there was a statistically significant correlation between the TCRbeta/gamma VF and the hprt exon 2/3 deletion frequency in PBL DNA from the same donors. These results show that the TCRbeta/gamma VF in healthy individuals changes with age and correlates with the frequency of hprt exon 2/3 deletion, another marker of aberrant V(D)J recombination in T-cells. However, no effect of smoking or present or previous exposure to genotoxic agents on TCRbeta/gamma VF was observed in this study. Thus, further studies are needed to prove the utility of TCRbeta/gamma gene rearrangement as a marker of genotoxic exposure.

DNA double-strand break repair, DNA-PK, and DNA ligases in two human squamous carcinoma cell lines with different radiosensitivity.

PURPOSE: Variation in sensitivity to radiotherapy among tumors has been related to the capacity of cells to repair radiation-induced DNA double-strand breaks (DSBs). DNA-dependent protein kinase (DNA-PK) and DNA ligases may affect DNA dsb rejoining. This study was performed to compare rate of rejoining of radiation-induced DSBs, DNA-PK, and DNA ligase activities in two human squamous carcinoma cell lines with different sensitivity to ionizing radiation. METHODS AND MATERIALS: Cell survival of two human squamous carcinoma cell lines, UM-SCC-1 and UM-SCC-14A, was determined by an in vitro clonogenic assay. DSB rejoining was studied using pulsed field gel electrophoresis (PFGE). DNA-PK activity was determined using BIOTRAK DNA-PK enzyme assay system (Amersham). DNA ligase activity in crude cell extracts was measured using [5'-33P] Poly (dA) x (oligo (dT) as a substrate. Proteolytic degradation of proteins was analyzed by means of Western blotting. RESULTS: Applying the commonly used linear-quadratic equation to describe cell survival, S = e-alphaD-betaD2, the two cell lines roughly have the same alpha value (approximately 0.40 Gy(-1)) whereas the beta value was considerably higher in UM-SCC-14A (0.067 Gy(-2)+/-0.007 Gy(-2) [SEM]) as compared to UM-SCC-1 (0.013 Gy(-2)+/-0.004 Gy(-2) [SEM]). Furthermore, UM-SCC-1 was more proficient in rejoining of X-ray-induced DSBs as compared to UM-SCC-14A as quantified by PFGE. The constitutive level of DNA-PK activity was 1.6 times higher in UM-SCC-1 as compared to UM-SCC-14A ( < 0.05). The constitutive level of DNA ligase activity was similar in the two cell lines. CONCLUSIONS: The results suggest that the proficiency in rejoining of DSBs is associated with DNA-PK activity but not with total DNA ligase activity.

Proteolytic cleavage of HsRad51 during apoptosis.

The Rad51 gene of Saccharomyces cerevisiae is required for genetic recombination and recombinational repair of DNA strand breaks. In higher eukaryotes Rad51 is essential for embryonic development, and is involved in cell proliferation and DNA repair. Here we show that human Rad51 (HsRad51) is proteolytically cleaved during apoptosis in two T-lymphocyte cell lines, Jurkat and PFI-285. Apoptosis was induced by camptothecin or anti-Fas monoclonal antibody (anti-Fas mAb). HsRad51 was cleaved with similar kinetics as human poly(ADP-ribose) polymerase (HsPARP) after treatment with either agent. The time course of cleavage coincided with internucleosomal DNA fragmentation. The HsRad51 fragments observed in apoptotic cells were identical to those generated from in vitro translated (IVT) HsRad51 exposed to activated Jurkat S-100 extract in a cell-free system. In each case, cleavage of HsRad51 was abolished by acetyl-Asp-Glu-Val-Asp-aldehyde (Ac-DEVD-CHO). However, cleavage of IVT HsRad51 could not be demonstrated using purified caspase-2, -3 or -6 to -10, and the identity of the responsible protease thus remains to be determined. In summary, we have shown that HsRad51 belongs to a group of repair proteins, including PARP and DNA-dependent protein kinase, which are specifically cleaved during the execution phase of apoptosis.

Variations in the frequency of T-cell receptor beta/gamma-interlocus recombination in long-term cultures of non-irradiated and X- and gamma-irradiated human lymphocytes.

PURPOSE: The increased level of illegitimate V(D)J recombination at the T-cell receptor (TCR) loci in lymphoid tumours as well as in T lymphocytes of ataxia telangiectasia patients and humans exposed to carcinogens in vivo suggest that site-specific interlocus recombination events could serve as markers of genomic instability and early genetic changes associated with carcinogenesis. The purpose of this study was to investigate the ability of ionizing radiation to induce TCRbeta/gamma-interlocus rearrangements in human lymphocytes in vitro. MATERIALS AND METHODS: Peripheral blood lymphocytes (PBL) from two healthy donors were exposed to 3 Gy of either X- or gamma-irradiation in vitro. Growth factor-stimulated cell cultures were established, and cell samples for DNA extraction were taken immediately after exposure and at several time points during long-term growth. A PCR-based method was used to measure the frequency of variant cells with Vgamma-Jbeta1 TCR rearrangements. RESULTS: The frequency of TCRbeta/gamma-variant cells was not significantly different in the irradiated and control cultures at any time studied up to 55 days after PHA-stimulation, indicatin that V(D)J-mediated Vgamma-Jbeta1 rearrangement is not induced by X- or gamma-irradiation under these conditions. However, in both irradiated and non-irradiated cultures, the frequency of TCRbeta/gamma variants increased approximately fourfold after mitogen stimulation, from a normal background level of 0.3-0.4 x 10(-5) to 1.3-1.6 x 10(-5) at days 4-9. These levels then gradually declined during prolonged cultivation, and after 2-4 weeks the frequency of variant cells was below the detection limit ( < 0.13 x 10(-5)). CONCLUSIONS: These results provide no evidence that TCRbeta/gamma gene rearrangements can be induced by X- or gamma-irradiation in vitro. However, in contrast with cells with normal TCR receptors, TCRbeta/gamma-variant cells display a relative growth advantage for 1-2 weeks, followed by gradual loss of proliferative capacity. Eventually, they are eliminated from the cell population or outnumbered by cells with normal TCR. If there are similar differences in vivo between cells with hybrid and normal TCR, this may explain the previously reported time- and season-dependent changes in the frequency of cells with hybrid TCR in occupationally exposed populations and individuals receiving cytostatic treatment.

Frequency and cell specificity of T-cell receptor interlocus recombination in human cells.

Immunoglobulin and T-cell receptor (TCR) genes are assembled by a site-specific rearrangement known as V(D)J [variable-(diversity)-joining] recombination. These rearrangements occur normally in pre-B- and pre-T-cells using signal sequences adjacent to coding exons for immunoglobulin and TCR genes, respectively. However, aberrant recombination may result in the generation of hybrid TCR genes by joining of TCR-beta with TCR-gamma specific sequences. Such hybrid TCR genes occur at a low frequency in peripheral blood lymphocytes (PBL) of healthy individuals, and can be detected by PCR amplification. We have determined the in vivo frequency of hybrid V gamma-J beta 1 TCR (hybrid TCR) genes in lymphocyte DNA from 12 healthy individuals. The average frequency was found to be 5.83 in 0.75 x 10(6) PBL, with a threefold difference between the highest and lowest individual value. The presence of similar TCR gene rearrangements in individual samples suggests that T-cells with a hybrid TCR gene are capable of clonal expansion in vivo. The individual hybrid TCR gene frequency remained relatively constant during 72 hours of in vitro cultivation. In long-term culture, the frequency gradually decreased, and after 28 days no hybrid TCR genes were detectable in lymphocyte DNA. These results show that T-cells with a hybrid TCR gene are able to respond to mitogen stimulation in vitro, and may have a proliferative disadvantage or are selected against during prolonged in vitro cultivation. No hybrid TCR genes were detected in ten proliferating T-cell clones, indicating that the rate of hybrid TCR gene formation is < 2.0 x 10(-8) per cell per cell division. No hybrid TCR genes were detected in DNA from B-lymphocytes, sperm, granulocytes, fibroblasts, keratinocytes, and three B-lymphoblastoid ataxia telangiectasia cell lines. In agreement with previous reports, the frequency of hybrid TCR genes in peripheral blood DNA from two ataxia telangiectasia patients was found to be more than 15-fold higher than in lymphocytes from normal individuals. These data show that formation of hybrid TCR genes is restricted to T-cells in vivo, and occurs at a very low frequency, if at all, in proliferating T-cells in vitro, and with an increased frequency in patients with ataxia telangiectasia.

Expression of the human RAD51 gene during the cell cycle in primary human peripheral blood lymphocytes.

The S. cerevisiae RAD51 gene product exerts important functions in meiotic and mitotic recombination, as well as in the repair of DNA double-strand breaks. We have studied the expression of the human RAD51 (HsRAD51) gene in primary human peripheral blood lymphocytes (PBLs). The HsRAD51 mRNA level increased three fold in mitogen stimulated PBLs, with a peak in the late S phase. A five fold increase of HsRAD51 protein levels was observed in late G2. Specific inhibition of DNA synthesis with aphidicolin did not block the induction of the HsRAD51 protein, indicating that HsRAD51 expression is independent of DNA replication. In contrast, after inhibition of RNA synthesis with actinomycin D and protein synthesis with cycloheximide, the HsRAD51 protein level decreased rapidly. Taken together, these results indicate that the HsRAD51 gene is transcriptionally regulated in human PBLs, and exerts its function during the late S and G2 phases of the cell cycle.

Spontaneous length variation in microsatellite DNA from human T-cell clones.

Recently, much interest has been focused on instability of microsatellite DNA sequences such as di- and tri-nucleotide repeats in human cancers. Certain tumors show an increased frequency of mutation leading to repeat length variation at microsatellite loci, and it is thought that such instability may be a marker for the transformed phenotype. However, the spontaneous frequency by which repetitive DNA such as CA-repeats undergoes size changes in normal human somatic cells is not known. Therefore, it is not possible to decide if there is an increase in the frequency of microsatellite mutation in specific tumors or if the change observed simply reflects the frequency of microsatellite mutation in the cell population from which the tumor originates. To investigate this we have established panels of T-lymphocyte clones from 28 healthy males and determined the spontaneous length variations at three CA-repeat markers that are often used to investigate satellite instability: D2S123, D9S180, and D10S197. We found 3 T-cell clones with altered microsatellite size in a total of 178. This corresponds to a background frequency of 3 somatic microsatellite mutations in 1,028 alleles studied, i.e., 2.9 x 10(-3). This frequency is comparable to that found in many tumors of the breast, brain, ovary, and skin but is considerably lower than the frequency of microsatellite mutation in tumors related to hereditary non-polyposis colorectal cancer.

The human RAD51/RecA homologue gene is not a candidate gene for Bloom's syndrome.

The human gene HSRAD51/RecA homologue has been investigated as a possible candidate gene involved in Bloom's syndrome. No mutations were found in the cDNA isolated from three different Bloom's syndrome cell lines, thus excluding the possibility that HSRAD51 is directly involved in the syndrome. Other possible candidates are discussed.

Mutations induced in the hypoxanthine phosphoribosyl transferase gene by three urban air pollutants: acetaldehyde, benzo[a]pyrene diolepoxide, and ethylene oxide.

Provisional mutational spectra at the hypoxanthine phosphoribosyl transferase (HPRT) locus in vitro have been worked out for acetaldehyde (AA) and benzo[a]pyrene diolepoxide (BPDE) in human (T)-lymphocytes and for ethylene oxide (EtO) in human diploid fibroblasts using Southern blotting and polymerase chain reaction (PCR)-based DNA sequencing techniques. The results indicate that large genomic deletions are the predominating hprt mutations caused by AA and EO, whereas BPDE induces point mutations that are mainly GC > TA transversions. The mutational spectra induced by the three agents are clearly different from the background spectrum in human T-cells. Thus, the hprt locus is a useful target for the study of chemical-specific mutational events that may help identify causes of background mutation in human cells in vivo.

Alzheimer's disease: molecular genetics and transgenic animal models.

Disease-causing mutations in the amyloid precursor protein (APP) gene have been found on chromosome 21 during the last 2 years in some early onset Alzheimer's disease (AD) families. Genetic evidence shows that other genes than the APP are also involved in the aetiology of AD. Linkage to a loci on chromosome 14 has been found in early onset disease. The identification of APP mutation has led to the realization that APP mismetabolism is a central event in the aetiology and pathogenesis of the disease. Experiments to test this in transgenic mice have so far met with little success. There are many possible explanations for the problems to generate transgenic mice. These include the possibilities that mice are incapable of developing AD for reasons dependent on their APP sequence; and that appropriate regulation of APP gene is required for pathology to develop. Current attempts that seem promising to model the disease pathology are the use of homologous recombination to insert the pathogenic mutation and transfection of YACs into transgenic animals.

Duplication in the hypoxanthine phosphoribosyl-transferase gene caused by Alu-Alu recombination in a patient with Lesch Nyhan syndrome.

We have determined the structure, at the nucleotide sequence level, of a duplication in the hprt gene in a patient with Lesch-Nyhan syndrome (LN). The duplication extends over exons 7 and 8 and approximately 1.8 kb of the surrounding hprt sequence. The duplication junction is localized within two Alu sequences and has apparently been generated by unequal homologous recombination. This is the second reported case of a partial duplication of the hprt gene in an LN patient, and the first that involves an Alu-Alu recombination.

Missense mutations and evolutionary conserved amino acids at the human hypoxanthine phosphoribosyl-transferase locus.

Molecular characterization of in vivo mutation at the human hypoxanthine phosphoribosyltransferase (hprt) locus has revealed a broad spectrum of mutation, both with regard to germ-line mutation in Lesch-Nyhan and gout patients, and somatic mutation in 6-thioguanine resistant T-lymphocytes from healthy individuals. The pattern of missense mutation shows a non-random distribution with a preferential location to codons for amino acids which are identical in human and the two parasites Schistosoma mansoni and Plasmodium falciparum. Although these 'evolutionary conserved' amino acids account for only 32% of the amino acids in the human hprt protein, they are involved in 76% of the missense mutations at the hprt locus in human T-lymphocytes, 67% in Lesch-Nyhan patients (with severe hprt-deficiency), but only 43% in gout patients (with partial hprt deficiency). This observation supports the notion that evolutionary conserved amino acids constitute functionally important sites in the hprt enzyme, and missense mutations affecting these amino acids will often lead to complete loss of enzyme activity. Substitutions of 'non-conserved' amino acids cause less severe hprt-deficiency (as seen in the gout patients), or may even escape clinical diagnosis. These considerations are important for the understanding of structure-activity relationships in the hprt protein, possible differences between hprt mutational spectra in germ-line and somatic cells, and the mutational spectra induced by specific exogeneous mutagens.