Translocation of unstable heterochromatin as the mechanism of sister chromatid exchange formation: a proposed hypothesis

The phenomenon of sister chromatid exchange has remained an enigma in that the actual mechanism for its formation has never been elucidated. It has long been suspected that the process involves some form of breakage and rejoining of DNA, but that hypothesis has never been proved. Recent work in this laboratory using cells from a premature aging disorder (Werner's syndrome) has promulgated the hypothesis that heterochromatin may not be an integral structure of chromosomes, but rather serves as a surface feature or covering. Furthermore, heterochromatin in Werner's syndrome chromosomes was found to be unstable and easily sloughed-off the chromosome surface. In this investigation evidence is presented which shows that incorporation of bromodeoxyuridine into DNA causes instability in the purported heterochromatin covering, resulting in translocation of segments of heterochromatin from the unifilarly-substituted chromatid to the bifilarly-substituted sister chromatid. Such translocation may represent the long-elusive mechanism of sister chromatid exchange formation.     

Sgs1, a Homologue of the Bloom''s and Werner''s Syndrome Genes, Is Required for Maintenance of Genome Stability in Saccharomyces Cerevisiae

The Saccharomyces cerevisiae SGS1 gene is homologous to Escherichia coli RecQ and the human BLM and WRN proteins that are defective in the cancer-prone disorder Bloom's syndrome and the premature aging disorder Werner's syndrome, respectively. While recQ mutants are deficient in conjugational recombination and DNA repair, Bloom's syndrome cell lines show hyperrecombination. Bloom's and Werner's syndrome cell lines both exhibit chromosomal instability. sgs1Δ strains show mitotic hyperrecombination, as do Bloom's cells. This was manifested as an increase in the frequency of interchromosomal homologous recombination, intrachromosomal excision recombination, and ectopic recombination. Hyperrecombination was partially independent of both RAD52 and RAD1. Meiotic recombination was not increased in sgs1Δ mutants, although meiosis I chromosome missegregation has been shown to be elevated. sgs1Δ suppresses the slow growth of a top3Δ strain lacking topoisomerase III. Although there was an increase in subtelomeric Y' instability in sgs1Δ strains due to hyperrecombination, no evidence was found for an increase in the instability of terminal telomeric sequences in a top3Δ or a sgs1Δ strain. This contrasts with the telomere maintenance defects of Werner's patients. We conclude that the SGS1 gene product is involved in the maintenance of genome stability in S. cerevisiae.     

Chromosomal abnormalities in lymphocytes from a patient with Werner's syndrome in intact culture and after treatment with halogenated analogs of thymidine

Four balanced chromosomal translocation, deletion of chromosome 15, and a break in chromosome 11 were detected in 100 G-banded metaphases of cultured lymphocytes of a patient with Werner's syndrome. We observed aneuploidy that included both trisomies and monosomies for various chromosomes. Halogenated analogs of thymidine in low doses increased significantly the incidence of chromosome aberrations accompanied by fragments. 5-Iododeoxyuridine induced lesions in centromeric regions of B-group chromosomes in 44.4% of all the cases of breaks. A hypothesis is proposed about the existence of a special mechanism for genetic control in changes in the cell nucleus and mitotic chromosome transformation. This mechanism can be manifested after the application of halogenated analogs of thymidine. The mutation involved in Werner's syndrome is presumably related to this mode of genetic control.     

'Sloughing-off' of heterochromatin in Werner's syndrome cells during high-temperature phosphate incubation

Previous investigations of cells undergoing rapid division revealed the presence of heterochromatic 'dots' in chromosomes as well as numerous chromocentres in interphase nuclei. Such structures were seen in human embryonic cells, as well as cells from organisms capable of regeneration, and cells from various malignancies. Cells with a reduced capacity for reproduction were found to be virtually devoid of nuclear chromocentres and chromosome dots after incubation in phosphate buffer at high temperature. The lack of heterochromatin in such cells (Werner's syndrome) thereby explained their reduced capacity for cell division and the resultant rapid rate of aging in individuals afflicted. Re-examination of such slides containing these cells revealed that chromocentres and chromosome dots were present initially, but the incubation process resulted in a 'sloughing-off' of such structures. The incubation process left these heterochromatic structures intact in malignant and control cells, inferring a link between cell proliferation and stable intact heterochromatin. These findings implicate heterochromatin as the object of the purported chromosomal instability factor characteristic of Werner's syndrome. The loss of heterochromatin did not result in chromosome breakage, suggesting that heterochromatin may not be an integral part of chromosome structure, but rather a surface feature or covering.     

Elevation of urinary hyaluronic acid in Werner's syndrome and progeria

Werner's syndrome and Hutchinson-Gilford progeria syndrome (progeria) are human genetic diseases which may serve as models for the study of premature aging. The basic defects underlying these diseases are unknown. An abnormally high level of urinary hyaluronic acid (HA) excretion has been previously reported in several Werner's and one progeria subject, all from Japan. To determine if a high HA level is a reliable marker for these diseases, we quantitated the urinary excretion of HA in three progeria subjects, one subject with an atypical progeroid syndrome, and a Werner's syndrome subject. Compared to controls, the total urinary HA was found to be markedly increased in the three progeria samples and in the Werner's syndrome sample. These findings support the previous observations indicating elevated HA may be a specific marker for these diseases.     

Clonal structural chromosomal rearrangements in lymphocytes of four patients with Werner's syndrome

Multiple numerical and structural chromosome abnormalities were found in cultured lymphocytes of four patients with Werner's syndrome. The proportion of metaphases with structural and/or numerical aberrations varied from 30 to 44% and several of them were clonal. These results confirm definitively that Werner's syndrome is a chromosome rearrangement syndrome and that these non-constitutional chromosome changes are not exclusive of cultured fibroblasts but present also in lymphocytes.     

Premature aging in RecQ helicase-deficient human syndromes

The RecQ family of DNA helicases have potential roles in DNA repair, replication and/or recombination pathways. In humans, a defect in the RecQ family helicases encoded by the BLM, WRN and RECQ4 genes gives rise to Bloom's (BS), Werner's (WS) and Rothmund-Thomson (RTS) syndromes, respectively. These disorders are associated with cancer predisposition and/or premature aging. In Bloom's syndrome, affected individuals are predisposed to many types of cancer at an early age. Werner's syndrome is a premature aging disorder with a complex phenotype, which includes many age-related disorders that develop from puberty, including greying and thinning of the hair, bilateral cataract formation, type II diabetes mellitus, osteoporosis and atherosclerosis. The phenotype of Rothmund-Thomson syndrome patients also consists of some features associated with premature aging, as well as predispositon to certain cancers. Here, we discuss the molecular basis of these RecQ helicase-deficient disorders.     

The Marfan syndrome locus: confirmation of assignment to chromosome 15 and identification of tightly linked markers at 15q15-q21.3

The Marfan syndrome is a common autosomal dominant disorder of connective tissue. Despite many years of intensive investigation, the primary genetic defect has not yet been identified. Reverse genetic methods, targeted at mapping this disease gene, have resulted in an initial report of linkage of the genetic locus for the Marfan phenotype in Finnish families to two polymorphic markers on chromosome 15. We have investigated four large multiplex American families with classic Marfan syndrome using standard genetic linkage methods. Our data confirm the assignment of the Marfan syndrome gene to chromosome 15, but establish a more centromeric location (defined by markers D15S25 and D15S1) as the most probable site for the genetic defect (lod score = 12.1, theta = 0.00). These data should facilitate identification and characterization of the Marfan syndrome gene and, in selected families, have immediate application to diagnosis of equivocal cases or prenatal counseling.     

Limb mammary syndrome: a new genetic disorder with mammary hypoplasia, ectrodactyly, and other Hand/Foot anomalies maps to human chromosome 3q27.

We report on a large Dutch family with a syndrome characterized by severe hand and/or foot anomalies, and hypoplasia/aplasia of the mammary gland and nipple. Less frequent findings include lacrimal-duct atresia, nail dysplasia, hypohydrosis, hypodontia, and cleft palate with or without bifid uvula. This combination of symptoms has not been reported previously, although there is overlap with the ulnar mammary syndrome (UMS) and with ectrodactyly, ectodermal dysplasia, and clefting syndrome. Allelism with UMS and other related syndromes was excluded by linkage studies with markers from the relevant chromosomal regions. A genomewide screening with polymorphic markers allowed the localization of the genetic defect to the subtelomeric region of chromosome 3q. Haplotype analysis reduced the critical region to a 3-cM interval of chromosome 3q27. This chromosomal segment has not been implicated previously in disorders with defective development of limbs and/or mammary tissue. Therefore, we propose to call this apparently new disorder "limb mammary syndrome" (LMS). The SOX2 gene at 3q27 might be considered an excellent candidate gene for LMS because the corresponding protein stimulates expression of FGF4, an important signaling molecule during limb outgrowth and development. However, no mutations were found in the SOX2 open reading frame, thus excluding its involvement in LMS.     

Genetic modulation of the senescent phenotype in Homo sapiens

While it is important to search for unifying mechanisms of aging among a variety of model systems, evolutionary arguments suggest that the pathophysiological details of senescence may be, to some extent, species specific. Moreover, in species that are characterized by extensive genetic heterogeneity, such as our own, one is likely to find kindreds with both "private" and "public" markers of aging. Crude estimates of the number of loci with the potential to modulate aspects of the senescent phenotype of man suggest that thousands of genes could be involved. No single locus appears to modulate all features. Some affect predominantly a single aspect ("unimodal progeroid syndromes"); familial Alzheimer's disease is discussed as a prototype. Linkage studies indicate genetic heterogeneity for autosomal dominant forms of the disease. Some loci affect multiple aspects of the phenotype ("segmental progeroid disorders"); the prototype is Werner's syndrome, an autosomal recessive. Cells from homozygotes behave like mutator strains and undergo accelerated senescence in vitro. Elucidation of the biochemical genetic basis of such abiotrophic disorders may shed light on specific aging processes in man.     

Clinical impact of molecular diagnostics in endocrinology. Polymorphisms,mutations and DNA technologies

Genetic defects in genes encoding hormones, hormone receptors or polypeptides of the signaling pathways usually cause complex disease manifestations characterized by the involvement of several tissues and variable expression. Genetic aberrations, like chromosome aneuploidy, gene translocations or mutations in key regulatory proteins (even if not directly affecting genes of the endocrine system) often lead to clinical symptoms, including central endocrine functions like sexual differentiation or metabolic disturbances, like diabetes mellitus. But also minor genetic alterations like point mutations can affect the function of gene products to cause endocrine diseases. If the underlying molecular defects of endocrinopathies are known, direct molecular diagnosis can be performed. This is particularly useful if it helps to solve difficult differential diagnosis problems or if there exist effective preventive therapeutic options. The present paper presents examples for endocrine diseases in which molecular testing significantly increases the specificity and sensitivity of diagnostics and demonstrates the benefits for the patients and the healthcare system. In multiple endocrine neoplasia type 2, an unambiguous identification of gene carriers in affected families can be achieved by genetic testing. As a preventive measure to avoid medullary thyroid carcinoma, prophylactic thyroidectomy is recommended for individuals carrying the disease causing mutation. In adrenogenital syndrome, sequence analysis of the steroid 21-hydroxylase gene has become an important tool to confirm or exclude suspected late-onset forms of the disease, where hormone measurements are not informative. The major benefit, however, lies in identifying heterozygous carriers and providing a reliable prenatal test for couples carrying a defect in the 21-hydroxylase gene. Today, prenatal treatment with dexamethasone, which prevents the virilization in female fetuses, should always be based on results from molecular diagnosis performed from chorionic villus samples.     

Elevated incidence of loss of heterozygosity (LOH) in an sgs1 mutant of Saccharomyces cerevisiae: roles of yeast RecQ helicase in suppression of aneuploidy,interchromosomal rearrangement,and the simultaneous incidence of both events during mitotic growth

The SGS1 gene of Saccharomyces cerevisiae is a member of the RecQ helicase family, which includes the human BLM, WRN and RECQL4 genes responsible for Bloom and Werner's syndrome and Rothmund-Thomson syndrome, respectively. Cells defective in any of these genes exhibit a higher incidence of genome instability. We previously demonstrated that various genetic alterations were detectable as events leading to loss of heterozygosity (LOH) in S. cerevisiae diploid cells, utilizing a hemizygous URA3 marker placed at the center of the right arm of chromosome III. Analyses of chromosome structure in LOH clones by pulse field gel electrophoresis (PFGE) and PCR, coupled with a genetic method, allow identification of genetic alterations leading to the LOH. Such alterations include chromosome loss, chromosomal rearrangements at various locations and intragenic mutation. In this work, we have investigated the LOH events occurring in cells lacking the SGS1 gene. The frequencies of all types of LOH events, excluding intragenic mutation, were increased in sgs1 null mutants as compared to the wild-type cells. Loss of chromosome III and chromosomal rearrangements were increased 13- and 17-fold, respectively. Further classification of the chromosomal rearrangements confirmed that two kinds of events were especially increased in the sgs1 mutants: (1) ectopic recombination between chromosomes, that is, unequal crossing over and translocation (46-fold); and (2) allelic crossing over associated with chromosome loss (40-fold). These findings raise the possibility that the Sgs1 protein is involved in the processing of recombination intermediates as well as in the prevention of recombination repair during chromosome DNA replication. On the other hand, intrachromosomal deletions between MAT and HMR were increased only slightly (2.9-fold) in the sgs1 mutants. These results clearly indicate that defects in the SGS1 gene function lead to an elevated incidence of LOH in multiple ways, including chromosome loss and interchromosomal rearrangements, but not intrachromosomal deletion.     

Effects of radical-scavenging enzymes and reduced oxygen exposure on growth and chromosome abnormalities of Werner syndrome cultured skin fibroblasts

Summary Two sister strains of skin fibroblast-like (FL) cells from a patient with Werner's syndrome (adult progeria) were grown in regular tissue culture medium or medium supplemented with the radical-scavenging enzymes superoxide dismutase and catalase. Differences in proliferative potential were shown to be due to strain variation rather than to treatment with the enzymes. Two independent strains were also grown in ambient (18%) or reduced (1%) concentrations of oxygen. All cultures (untreated, treated with enzymes, treated with reduced oxygen exposure) displayed the limited in vitro growth potential and cytogenetic abnormality characteristic of Werner's syndrome FL cells; thus the underlying defect in Werner's syndrome does not appear to be related to defective protection against or repair of damage by free radicals.     

The Rb/E2F pathway and Ras activation regulate RecQ helicase gene expression

Disruption of the Rb (retinoblastoma protein)/E2F cell-cycle pathway and Ras activation are two of the most frequent events in cancer, and both of these mutations place oncogenic stress on cells to increase DNA replication. In the present study, we demonstrate that these mutations have an additive effect on induction of members of the RecQ DNA helicase family. RecQ activity is important for genomic stability, initiation of DNA replication and telomere maintenance, and mutation of the BLM (Bloom's syndrome gene), WRN (Werner's syndrome gene) or RECQL4 (Rothmund-Thomson syndrome gene) family members leads to premature aging syndromes characterized by genetic instability and telomere loss. RecQ family members are frequently overexpressed in cancers, and overexpression of BLM has been shown to cause telomere elongation. Concomitant with induction of RecQ genes in response to Rb family mutation and Ras activation, we show an increase in the number of telomeric repeats. We suggest that this induction of RecQ genes in response to common oncogenic mutations may explain the up-regulation of the genes seen in cancers, and it may provide a means for transformed cells to respond to an increased demand for DNA replication.     

Clonal structural chromosomal rearrangements in primary fibroblast cultures and in lymphocytes of patients with Werner's syndrome

Summary The cytogenetics of six cases of adult progeria (Werner's syndrome) from three Sardinian families were investigated. The overall increased incidence of chromosome breakage found in cultured lymphocytes and fibroblasts seems to be age-dependent. The occurrence of clonal variegated translocation mosaicism, previously found by other authors in fibroblast cell lines derived from Werner patients was demonstrated also in fibroblasts analyzed in situ on the outgrowth halos from primary skin explants; a strong indication that these aberrations are present in the in vivo precursors. The same type of clonal structural aberration was found for the first time also in 72h-cultured lymphocytes. These findings demonstrate that Werner's syndrome is indeed a further example of a chromosome rearrangement syndrome.     

Telomeres, hidden mosaicism, loss of heterozygosity, and complex genetic traits

Telomeres appear to function as an endogenous timing mechanism in human beings. Telomere attrition not only provides a satisfactory explanation for some aspects of aging, it might also resolve enigmatic features of complex genetic traits that are age-dependent. If, with the passage of time, telomere attrition in human beings leads to genomic instability and particularly the loss of chromosomes, then the age dependency of phenotypic expressions of complex genetic traits might result from the temporal loss of heterozygosity and the consequent expression of disease-causing genes. In this way, telomere attrition might play a role not only in aging, but also in the diverse expression of complex genetic traits, such as essential hypertension, non-insulin-dependent diabetes mellitus, atherosclerosis, and cancer.     

Impaired S-phase transit of Werner syndrome cells expressed in lymphoblastoid cell lines

The clinical phenotype of Werner's syndrome (WS) includes short stature, premature cataracts, skin atrophy, osteoporosis, graying and loss of hair, neoplasia, diabetes mellitus, and arteriosclerosis. Cultured cells from patients with this autosomal recessive disorder exhibit chromosomal instability and a markedly reduced replicative lifespan and growth rate. To elucidate the cell cycle alterations associated with the growth deficit, we continuously labeled lymphoid cell lines from five WS patients and from four healthy adult controls with 5-bromodeoxyuridine. Bivariate Hoechst 33258/ethidium bromide flow cytometry revealed a 2.4-h prolongation in the minimal duration of the S phase of WS cells (P less than 0.005). Moreover, the fraction of proliferating cells irreversibly arrested in the S phase (5.4% vs 1.4% in controls) was significantly elevated in WS (P less than 0.001). Other cell cycle compartments were not significantly affected in WS cell lines. As a partial test of the hypothesis that the WS phenotype is due to a defect in DNA topoisomerase I (topo I) or DNA topoisomerase II (topo II) we exposed lymphoid cells from a healthy control to the topo I inhibitor camptothecin or to the topo II inhibitor 4'-(9-acridinylamino)methanesulfon-m-anisidine. The cell kinetic alterations elicited by these compounds differed from that exhibited by untreated WS patients. Thus, a primary defect in topo I or II is unlikely in WS. Our cell cycle results, however, provide important evidence that the biochemical genetic lesion is in fact expressed in lymphoblastoid cell lines, the most readily available cells from such subjects.     

Down syndrome: pathogenesis,radioresistant DNA synthesis and chromosomal instability

Down syndrome (DS) is a frequent chromosomal aberration. Triplication of the fragment 21q22 of chromosome 21 is sufficient to cause the DS phenotype including immunodeficiency, premature aging, mental retardation, and an increased risk of leukemia. Chromosomal aberrations caused by X-ray irradiation were observed in DS lymphocytes and DS fibroblasts, but the correlation between chromosomal sensitivity, repair deficiency, and radioresistant DNA synthesis was not clear. Here some insight into the nature of this problem has been made. Besides, new arguments have been provided in favour of genetic heterogeneity of this genetic disorder.     

Age dymamics of stable chromosome aberration frequency in humans with natural and pathological senescence

The age dynamics of stable chromosome aberration (SCA) frequency was analysed by fluorescent in situ hybridization (FISH) in human blood lymphocytes derived from donors, irradiated by low doses of ionizing radiation (Chernobyl clean-up workers, nuclear weapon testers, etc.) and patients with hereditary premature aging--Werner's syndrome and Hutchinson-Gilford's syndrome. It was found that the level of SCA was age-dependent and increased in irradiated persons. So, the SCA level may be really an index of a so-called "radiation senescence", and may show a real biological age of irradiated persons. The patients with Werner's syndrome demonstrate increased SCA level in blood lymphocytes, corresponding to the premature aging of the organisms. But in the case of another form of premature aging--Hutchinson--Gilford's syndrome-- no rise of SCA level was found. Some possible reasons of such results are discussed.     

The impact of imprinting: Prader-Willi syndrome resulting from chromosome translocation, recombination, and nondisjunction.

Prader-Willi syndrome (PWS) is most often the result of a deletion of bands q11.2-q13 of the paternally derived chromosome 15, but it also occurs either because of maternal uniparental disomy (UPD) of this region or, rarely, from a methylation imprinting defect. A significant number of cases are due to structural rearrangements of the pericentromeric region of chromosome 15. We report two cases of PWS with UPD in which there was a meiosis I nondisjunction error involving an altered chromosome 15 produced by both a translocation event between the heteromorphic satellite regions of chromosomes 14 and 15 and recombination. In both cases, high-resolution banding of the long arm was normal, and FISH of probes D15S11, SNRPN, D15S10, and GABRB3 indicated no loss of this material. Chromosome heteromorphism analysis showed that each patient had maternal heterodisomy of the chromosome 15 short arm, whereas PCR of microsatellites demonstrated allele-specific maternal isodisomy and heterodisomy of the long arm. SNRPN gene methylation analysis revealed only a maternal imprint in both patients. We suggest that the chromosome structural rearrangements, combined with recombination in these patients, disrupted normal segregation of an imprinted region, resulting in uniparental disomy and PWS.