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The article reports on the assignment of phosphotriesterase-related gene (PIER) to human chromosome band 10p12 by in situ hybridization. Phosphotriesterases comprise a family of prokaryotic enzymes that catalyse the hydrolysis of various phosphotriester molecules. Eukaryotic phosphotriesterases have not yet been identified but recently mouse and rat cDNAs encoding proteins with a high degree of homology to these enzymes have been characterised. Human genomic clones encoding a phosphotriesterase homologue were isolated.

Epidemiological investigations show that mosaic loss of chromosome Y (LOY) in leukocytes is associated with earlier mortality and morbidity from many diseases in men. LOY is the most common acquired mutation and is associated with aberrant clonal expansion of cells, yet it remains unclear whether this mosaicism exerts a direct physiological effect. We studied DNA and RNA from leukocytes in sorted- and single-cells in vivo and in vitro. DNA analyses of sorted cells showed that men diagnosed with Alzheimer's disease was primarily affected with LOY in NK cells whereas prostate cancer patients more frequently displayed LOY in CD4 + T cells and granulocytes. Moreover, bulk and single-cell RNA sequencing in leukocytes allowed scoring of LOY from mRNA data and confirmed considerable variation in the rate of LOY across individuals and cell types. LOY-associated transcriptional effect (LATE) was observed in ~ 500 autosomal genes showing dysregulation in leukocytes with LOY. The fraction of LATE genes within specific cell types was substantially larger than the fraction of LATE genes shared between different subsets of leukocytes, suggesting that LOY might have pleiotropic effects. LATE genes are involved in immune functions but also encode proteins with roles in other diverse biological processes. Our findings highlight a surprisingly broad role for chromosome Y, challenging the view of it as a "genetic wasteland", and support the hypothesis that altered immune function in leukocytes could be a mechanism linking LOY to increased risk for disease.

Carriers of cytogenetically similar, apparently balanced familial chromosome translocations not always exhibit the putative translocation-associated disease phenotype. Additional genetic defects, such as genomic imbalance at breakpoint regions or elsewhere in the genome, have been reported as the most plausible explanation.By means of comprehensive molecular and functional analyses, additional to careful dissection of the t(3;14)(q26.33;q12) breakpoints, we unveil a novel X-linked PGK1 mutation and examine the contribution of these to the extremely severe clinical phenotype characterized by hemolytic anemia and neuromyopathy.The 3q26.33 breakpoint is 40 kb from the 5' region of tetratricopeptide repeat domain 14 gene (TTC14), whereas the 14q12 breakpoint is within IVS6 of nucleotide-binding protein-like gene (NUBPL) that encodes a mitochondrial complex I assembly factor. Disruption of NUBPL in translocation carriers leads to a decrease in the corresponding mRNA accompanied by a decrease in protein level. Exclusion of pathogenic genomic imbalance and reassessment of familial clinical history indicate the existence of an additional causal genetic defect. Consequently, by WES a novel mutation, c.358G>A, p.E120K, in the X-linked phosphoglycerate kinase 1 (PGK1) was identified that segregates with the phenotype. Specific activity, kinetic properties, and thermal stability of this enzyme variant were severely affected. The novel PGK1 mutation is the primary genetic alteration underlying the reported phenotype as the translocation per se only results in a subclinical phenotype. Nevertheless, its co-inheritance presumably exacerbates PGK1-deficient phenotype, most likely due to a synergistic interaction of the affected genes both involved in cell energy supply.

Although the cause and outcome of mitotic catastrophe (MC) has been thoroughly investigated, precisely how the ensuing lethality is regulated during or following this process and what signals are involved remain unknown. Moreover, the mechanism of the decision of cell death modalities following MC is still not well characterised. We demonstrate here a crucial role of the γH2AX-ATM-p53 pathway in the regulation of the apoptotic outcome of MC resulting from cells entering mitosis with damaged DNA. In addition to p53 deficiency, the depletion of ATM (ataxia telangiectasia mutated), but not ATR (ataxia telangiectasia and Rad3-related protein), protected against apoptosis and shifted cell death towards necrosis. Activation of this pathway is triggered by the augmented chromosomal damage acquired during anaphase in doxorubicin-treated cells lacking 14-3-3σ (also known as epithelial cell marker protein-1 or stratifin). Moreover, cells that enter mitosis with damaged DNA encounter segregation problems because of their abnormal chromosomes, leading to defects in mitotic exit, and they therefore accumulate in G1 phase. These multi- or micronucleated cells are prevented from cycling again in a p53- and p21-dependent manner, and subsequently die. Because increased chromosomal damage resulting in extensive H2AX phosphorylation appears to be a direct cause of catastrophic mitosis, our results describe a mechanism that involves generation of additional DNA damage during MC to eliminate chromosomally unstable cells.

To further explore the extent of structural large-scale variation in the human genome, we assessed copy number variations (CNVs) in a series of 71 healthy subjects from three ethnic groups. CNVs were analyzed using comparative genomic hybridization (CGH) to a BAC array covering the human genome, using DNA extracted from peripheral blood, thus avoiding any culture-induced rearrangements. By applying a newly developed computational algorithm based on Hidden Markov modeling, we identified 1,078 autosomal CNVs, including at least two neighboring/overlapping BACs, which represent 315 distinct regions. The average size of the sequence polymorphisms was approximately 350 kb and involved in total approximately 117 Mb or approximately 3.5% of the genome. Gains were about four times more common than deletions, and segmental duplications (SDs) were overrepresented, especially in larger deletion variants. This strengthens the notion that SDs often define hotspots of chromosomal rearrangements. Over 60% of the identified autosomal rearrangements match previously reported CNVs, recognized with various platforms. However, results from chromosome X do not agree well with the previously annotated CNVs. Furthermore, data from single BACs deviating in copy number suggest that our above estimate of total variation is conservative. This report contributes to the establishment of the common baseline for CNV, which is an important resource in human genetics.

Multiple regions on the chromosome arm 3p are frequently affected by loss of heterozygosity in human cancers. A candidate tumor suppressor gene is TMEM7, at 3p21.3, which encodes a transmembrane protein. TMEM7 is expressed specifically in the liver, and the encoded protein shares substantial sequence homology with human and mouse 28-kDa interferon-alpha (IFN-alpha) responsive protein. In investigation of the possible role of TMEM7 in development of hepatocellular carcinoma (HCC), we examined TMEM7 expression in 20 primary HCC and 18 HCC cell lines and found recurrent functional alterations. Although TMEM7 mRNA was expressed in normal hepatic cells, downregulation or inactivation of the gene was detected in 85% of primary HCC and 33% of HCC cell lines. To identify the mechanisms responsible, we examined genomic deletion and mutation, and also the effect of inhibitors of DNA methyltransferase and histone deacetylase on cells with low or no endogenous TMEM7 expression. Homozygous deletion of TMEM7 was not detected in 17 pairs of human HCC and corresponding noncancerous liver tissues, nor in any of the 18 HCC cell lines. TMEM7 mutation was not detected in the 18 HCC cell lines (low or normal TMEM7 expression). Treatment of two of six cell lines exhibiting downregulation or loss of TMEM7 with 5-aza-2'-deoxycytidine and trichostatin A yielded additive increase in TMEM7 expression, implicating aberrant DNA methylation and histone deacetylation in transcriptional silencing of this gene. Ectopic expression of TMEM7 in two TMEM7-deficient HCC lines suppressed cell proliferation, colony formation, and cell migration in vitro and reduced tumor formation in nude mice. Treatment of two highly invasive HCC cell lines with IFN-alpha for 7 days significantly increased TMEM7 expression and inhibited cell migration. These findings implicate loss of TMEM7 expression in hepatocarcinogenesis and suggest that modification of TMEM7 expression by IFN-alpha may have therapeutic relevance in a subset of HCC.

Transgenic mice were generated expressing NK1.1, an NK cell-associated receptor, under control of the human CD2 promoter. Unexpectedly, one of the founder lines, Tg66, showed a marked defect in thymic development characterized by disorganized architecture and small size. Mapping of the transgene insertion by fluorescence in situ hybridization revealed integration in chromosome 2, band G. Already from postnatal day 3, the thymic architecture was disturbed with a preferential loss of cortical thymic epithelial cells, a feature that became more pronounced over time. Compared with wild-type mice, total thymic cell numbers decreased dramatically between 10 and 20 days of age. Thymocytes isolated from adult Tg66 mice were predominantly immature double-negative cells, indicating a block in thymic development at an early stage of differentiation. Consequently, Tg66 mice had reduced numbers of peripheral CD4(+) and CD8(+) T cells. Bone marrow from Tg66 mice readily reconstituted thymi of irradiated wild-type as well as RAG-deficient mice. This indicates that the primary defect in Tg66 mice resided in nonhemopoietic stromal cells of the thymus. The phenotype is observed in mice heterozygous for the insertion and does not resemble any known mutations affecting thymic development. Preliminary studies in mice homozygous for transgene insertion reveal a more accelerated and pronounced phenotype suggesting a semidominant effect. The Tg66 mice may serve as a useful model to identify genes regulating thymic epithelial cell differentiation, thymic development, and function.

Chromosome deletions do abound in cancer and are detected in certain regions in a non-random manner. Although their relevance remains elusive, it is a general agreement that segmental losses provide the cell with selective growth advantage. Consequently these may contain genes and/or regulatory sequences that control normal growth and inhibit malignancy. We have developed a monochromosomal hybrid based experimental model for the generation and functional analysis of deletions, that is called "elimination test" (Et). Focused on human chromosome 3 - that was known to carry multiple 3p deletions - the Et was expected to restrict a 3p tumor suppressor region to a sufficiently small segment that permits the selection of a critically important candidate gene. Surprisingly, we detected three regions that were lost in all or majority of tumors: CER1 (3p21.3, Mb: 43.32-45.74), CER2 (3p22, Mb: 37.83-39.06) and FER (3p14.3-p21.2, Mb: 50.12-58.03). In contrast a 3q26-qter region (CRR) was regularly retained. CER1 - our main focus - contains multiple genes that may inhibit tumor growth, but 3 genes, RIS1, LF (LTF) and LIMD1 have already the necessary experimental support to be considered bona fide tumor suppressors. Tumor suppressor region borders display instability features including: (1) they break in evolution and in tumors, (2) they evolve horizontally, and (3) they are enriched with pseudogene insertions. The most remarkable features at the breakpoint cluster regions were segmental duplications that drive horizontal evolution and contribute to cancer associated instability.

Background: Euploid chromosome balance is vitally important for normal development, but is profoundly changed in many tumors. Is each tumor dependent on its own structurally and numerically changed chromosome complement that has evolved during its development and progression? We have previously shown that normal chromosome 3 transfer into the KH39 renal cell carcinoma line and into the Hone1 nasopharyngeal carcinoma line inhibited their tumorigenicity. The aim of the present study was to distinguish between a qualitative and a quantitative model of this suppression. According to the former, a damaged or deleted tumor suppressor gene would be restored by the transfer of a normal chromosome. If so, suppression would be released only when the corresponding sequences of the exogenous normal chromosome are lost or inactivated. According to the alternative quantitative model, the tumor cell would not tolerate an increased dosage of the relevant gene or segment. If so, either a normal cell derived, or, a tumor derived endogenous segment could be lost., Methods: Fluorescence in Situ Hybridization based methods, as well as analysis of polymorphic microsatellite markers were used to follow chromosome 3 constitution changes in monochromosomal hybrids., Results: In both tumor lines with introduced supernumerary chromosomes 3, the copy number of 3p21 or the entire 3p tended to fall back to the original level during both in vitro and in vivo growth. An exogenous, normal cell derived, or an endogenous, tumor derived, chromosome segment was lost with similar probability. Identification of the lost versus retained segments showed that the intolerance for increased copy number was particularly strong for 3p14-p21, and weaker for other 3p regions. Gains in copy number were, on the other hand, well tolerated in the long arm and particularly the 3q26-q27 region., Conclusion: The inability of the cell to tolerate an experimentally imposed gain in 3p14-p21 in contrast to the well tolerated gain in 3q26-q27 is consistent with the fact that the former is often deleted in human tumors, whereas the latter is frequently amplified. The findings emphasize the importance of even minor changes in copy number in seemingly unbalanced aneuploid tumors.

Traditionally, surveillance against cancer was thought of as mainly immunological. With the exception of tumors with a clear viral involvement, such as immunoblastomas (Epstein-Barr virus, EBV), cervical, anogenital, and skin carcinomas (HPV), and Kaposi's sarcoma (HHV-8) where the immune system is confronted with virally encoded, nonself targets, tumors with no viral involvement provide poor targets. Attempts to influence them by immunological means are akin to the breaking of tolerance. Robust nonimmunological surveillance mechanisms include DNA repair-based checkpoint functions, and the triggering of growth arrest and/or apoptosis pathways by DNA damage or by illegitimate oncogene activation (intracellular surveillance). There is emerging evidence for epigenetic surveillance, reflected in the stringency of imprinting. A fourth mechanism, intercellular surveillance, or microenvironmental control, is rapidly gaining momentum. It can be mediated by contactual controls or by differentiation-inducing signals. Somatic hybridization experiments have shown that tumorigenicity is usually suppressed in somatic hybrids between normal and malignant cells, as long as a fairly complete chromosome complement is maintained. Individual normal cell-derived chromosomes may have a similar suppressive effect. For example, genetic and molecular dissection of human 3p that shows frequent deletions in many human tumors has identified multiple tumor suppressor genes, which can inhibit both in vitro growth and in vivo tumorigenicity. In addition, five genes were found with an "asymmetric activity," capable of suppressing tumorigenicity, without affecting in vitro growth. These genes, LTF, L1MD1, HYAL1, HYAL2, and VHL, are of particular interest because they may be involved in microenvironmental control.

We examined chromosome 3 in 32 childhood acute lymphoblastic leukemia (ALL) bone marrow samples. Using interphase multipoint FISH (mp-FISH), which was developed by our group, with 42 chromosome 3-specific probes, we detected clonal chromosome 3 aberrations in 4 T-cell ALL (T-ALL) cases. Four out of seven T-ALL cases carried 3q trisomies. One T-ALL case carried either trisomy 3 (in 15% of the cells) or a 23-megabase (Mb) 3p13 approximately p12 deletion in a different subpopulation of cells of 32%. Another T-ALL case had either 3q trisomy in 11% or a 12-Mb 3p12 approximately p13 deletion in 19% of the cells. The deletions were overlapping. In both cases, the majority of the bone marrow cells (47 and 70%, respectively) were normal chromosome 3 disomics. The interstitial deletions detected harbor a known homozygous deletion region between 72.6 and 78.8 Mb, which has been described in lung and breast tumors and contains the DUTT1/ROBO1 tumor suppressor gene. These deletions detected by mp-FISH would have remained unnoticed by conventional cytogenetics and multiplex FISH, as well as by current methods based on total tumor DNA analysis such as comparative genomic hybridization (CGH), array CGH, and loss of heterozygosity (LOH).

Background: Recently, several high-resolution methods of chromosome analysis have been developed. It is important to compare these methods and to select reliable combinations of techniques to analyze complex chromosomal rearrangements in tumours. In this study we have compared array-CGH (comparative genomic hybridization) and multipoint FISH (mpFISH) for their ability to characterize complex rearrangements on human chromosome 3 (chr3) in tumour cell lines. We have used 179 BAC/PAC clones covering chr3 with an approximately 1 Mb resolution to analyze nine carcinoma lines. Chr3 was chosen for analysis, because of its frequent rearrangements in human solid tumours., Results: The ploidy of the tumour cell lines ranged from near-diploid to near-pentaploid. Chr3 locus copy number was assessed by interphase and metaphase mpFISH. Totally 53 chr3 fragments were identified having copy numbers from 0 to 14. MpFISH results from the BAC/PAC clones and array-CGH gave mainly corresponding results. Each copy number change on the array profile could be related to a specific chromosome aberration detected by metaphase mpFISH. The analysis of the correlation between real copy number from mpFISH and the average normalized inter-locus fluorescence ratio (ANILFR) value detected by array-CGH demonstrated that copy number is a linear function of parameters that include the variable, ANILFR, and two constants, ploidy and background normalized fluorescence ratio., Conclusion: In most cases, the changes in copy number seen on array-CGH profiles reflected cumulative chromosome rearrangements. Most of them stemmed from unbalanced translocations. Although our chr3 BAC/PAC array could identify single copy number changes even in pentaploid cells, mpFISH provided a more accurate analysis in the dissection of complex karyotypes at high ploidy levels.

We have applied a functional test for tumour antagonizing genes based on human chromosome 3 (chr3)-mouse fibrosarcoma A9 MCHs that were studied in vitro and after growth as tumours in severe combined immunodeficiency (SCID) mice. Previously, we reported that 9 out of the 36 SCID-tumours maintained the transferred chr3 ("chr3+" tumours), but lost the expression of the known human TSG fragile histidine triad gene (FHIT) in contrast to 14 other 3p-genes examined. Here we report the results of the duplex RT-PCR analysis of 9 "chr3+" tumours and 3 parental MCHs. We have examined the expression of 34 human 3p-genes from known cancer-related regions of instability, including 13 genes from CER1 defined by us previously at 3p21.33-p21.31 and 10 genes from the LUCA region at 3p21.31. We have found that in addition to FHIT, expression of the LTF gene from CER1 at 3p21.33-p21.31 was lost in all 9 tumours analyzed. The transcript of the solute carrier family 38 member 3 gene (SLC38A3) gene from LUCA region at 3p21.31 was not found in 8 and was greatly reduced in 1 out of these 9 tumours. Expression of the down-regulated in renal cell carcinoma gene (DRR1) gene at 3p14.2 was lost in 7 and down regulated in 2 "chr3+" tumours. In the SCID-tumour derived cell lines treatment with 5-aza-2'-deoxycytidine restored the mRNA expression of LTF, indicating the integrity of DNA sequences. Notably that transcription of the LTF and 2 flanking genes, LRRC2 and TMEM7, as well as transcription of the SLC38A3 gene, were also impaired in all 5 RCC cell lines analyzed. Our data indicate these genes as putative tumour suppressor genes.

We detected non-random 3p losses and 3q gains on well-determined regions in both murine and human tumors using a microcell hybrid-based model system called 'elimination test'. We suggest that these are general malignancy-associated aberrations not necessarily linked to a particular tissue of origin. To examine chromosome 3 abnormalities, in 28 childhood acute myeloid leukemia bone marrow samples, we performed interphase multipoint-fluorescence in situ hybridization using 84 chromosome 3-specific probes and detected clonal chromosome 3 aberrations in nine cases, which is of a higher frequency than the previously reported one. In 3/28 children, a chromosome 3 abnormality was detected which was not visible using conventional cytogenetic analysis. We did not detect any 3p deletion. Increased copy number of 3q was found in four cases with trisomy of whole chromosome 3 and one case with 3q tetrasomy (isodisomy). We identified rare structural rearrangements in childhood acute myeloblastic leukemia, involving 3q21 and 3q26 loci around RPN1 and MDS1/EVI1 respectively. The poor outcome in pediatric patients with 3q rearrangements appears to be quite uniform.

The virtually obligatory presence of the Philadelphia chromosome may suggest a causal homogeneity, but chronic myelogenous leukemia (CML) is a clinically heterogeneous disease. This may be a consequence of the variable BCR breakpoints on chromosome 22 and of nonrandom secondary chromosomal abnormalities. We present the case of a boy, age 12, investigated in blastic phase of CML. Karyotyping with conventional and multiplex fluorescence in situ hybridization (FISH and M-FISH) karyotyping, complemented with reverse transcriptase-polymerase chain reaction, identified a variant Philadelphia translocation t(9;14;22)(q34;q32;q11) involving a cryptic BCR/ABL fusion with formation of the p190(Bcr-Abl) oncoprotein. M-FISH revealed also an unbalanced jumping translocation of 17q11 approximately qter alternatively present on chromosomes 14 or 20, apparently hithertofore unreported in hematological malignancies. Another secondary aberration, dup(3)(q25q28), was revealed by multipoint interphase FISH (mpI-FISH). Gain of this region is known in adult hematological malignancies and solid tumors, suggesting its general involvement in tumor initiation or progression (or both), regardless of tissue origin.

Mutations in the known predisposing breast cancer genes BRCA1 and BRCA2 account for only a small proportion (<10%) of breast cancer families in the Stockholm region of Sweden. This study aims to identify novel predisposing genes in non-BRCA1/BRCA2 breast cancer families. We have employed comparative genomic hybridization (CGH) and loss of heterozygosity (LOH) data in combination with data from a recently carried out genome wide linkage scan, in an effort to identify chromosomal regions harboring potential breast cancer genes. CGH revealed loss of chromosome 17 and chromosome 6q to be a frequent event in high-risk breast cancer families, while gain of 8q was most prevalent in low-risk families. The loss of chromosome 17 was consistent with previous LOH studies and so this region was investigated further. Disease was shown to be linked to chromosome 17 in those families exhibiting loss of the chromosome in both CGH and LOH analyses. An overlapping region of linkage was determined to lie between markers D17S1294 and D17S1293, fine mapping of the region delineated a region between markers D17S1880 and D17S1293. Ten genes were determined to lie within this 1.5 Mb region and families were screened for germline mutations in these genes. In conclusion, we have investigated one possible small region on chromosome 17 for its involvement in high-risk non-BRCA1/BRCA2 breast cancer families. No predisposing mutations were identified in the 10 genes investigated in this study, however further analysis of chromosome 17 is warranted.