The structure, function and evolution of a complete human chromosome 8.

The complete assembly of each human chromosome is essential for understanding human biology and evolution1,2. Here we use complementary long-read sequencing technologies to complete the linear assembly of human chromosome 8. Our assembly resolves the sequence of five previously long-standing gaps, including a 2.08-Mb centromeric α-satellite array, a 644-kb copy number polymorphism in the ß-defensin gene cluster that is important for disease risk, and an 863-kb variable number tandem repeat at chromosome 8q21.2 that can function as a neocentromere. We show that the centromeric α-satellite array is generally methylated except for a 73-kb hypomethylated region of diverse higher-order α-satellites enriched with CENP-A nucleosomes, consistent with the location of the kinetochore. In addition, we confirm the overall organization and methylation pattern of the centromere in a diploid human genome. Using a dual long-read sequencing approach, we complete high-quality draft assemblies of the orthologous centromere from chromosome 8 in chimpanzee, orangutan and macaque to reconstruct its evolutionary history. Comparative and phylogenetic analyses show that the higher-order α-satellite structure evolved in the great ape ancestor with a layered symmetry, in which more ancient higher-order repeats locate peripherally to monomeric α-satellites. We estimate that the mutation rate of centromeric satellite DNA is accelerated by more than 2.2-fold compared to the unique portions of the genome, and this acceleration extends into the flanking sequence.

Mitomycin C-induced pairing of heterochromatin reflects initiation of DNA repair and chromatid exchange formation.

Chromatid interchanges induced by the DNA cross-linking agent mitomycin C (MMC) are over-represented in human chromosomes containing large heterochromatic regions. We found that nearly all exchange breakpoints of chromosome 9 are located within the paracentromeric heterochromatin and over 70% of exchanges involving chromosome 9 are between its homologues. We provide evidence that the required pairing of chromosome 9 heterochromatic regions occurs in G(0)/G(1) and S-phase cells as a result of an active cellular process initiated upon MMC treatment. By contrast, no pairing was observed for a euchromatic paracentromeric region of the equal-sized chromosome 8. The MMC-induced pairing of chromosome 9 heterochromatin is observed in a subset of cells; its percentage closely mimics the frequency of homologous interchanges found at metaphase. Moreover, the absence of pairing in cells derived from XPF patients correlates with an altered spectrum of MMC-induced exchanges. Together, the data suggest that the heterochromatin-specific pairing following MMC treatment reflects the initiation of DNA cross-link repair and the formation of exchanges.

The microcell mediated transfer of human chromosome 8 into highly metastatic rat liver cancer cell line C5F.

AIM: Our previous research on the surgical samples of primary liver cancer with CGH showed that the loss of human chromosome 8p had correlation with the metastatic phenotype of liver cancer. In order to seek the functional evidence that there could be a metastatsis suppressor gene(s) for liver cancer on human chromosome 8, we tried to transfer normal human chromosome 8 into rat liver cancer cell line C5F, which had high metastatic potential to lung. METHODS: Human chromosome 8 randomly marked with neo gene was introduced into C5F cell line by MMCT and positive microcell hybrids were screened by double selections of G418 and HAT. Single cell isolation cloning was applied to clone microcell hybrids. Finally, STS-PCR and WCP-FISH were used to confirm the introduction. RESULTS: Microcell hybrids resistant to HAT and G418 were obtained and 15 clones were obtained by single-cell isolation cloning. STS-PCR and WCP-FISH proved that human chromosome 8 had been successfully introduced into rat liver cancer cell line C5F. STS-PCR detected a random loss in the chromosome introduced and WCP-FISH found a consistent recombination of the introduced human chromosome with the rat chromosome. CONCLUSION: The successful introduction of human chromosome 8 into highly metastatic rat liver cancer cell line builds the basis for seeking functional evidence of a metastasis suppressor gene for liver cancer harboring on human chromosome 8 and its subsequent cloning.

Relations of the c-myc gene and chromosome 8 in non-small cell lung cancer: analysis by fluorescence in situ hybridization.

BACKGROUND: Amplification of the c-myc gene has been reported in non-small cell lung cancer (NSCLC). We investigated the c-myc gene amplification and the numerical aberration of chromosome 8 by dual color fluorescence in situ hybridization (FISH) to evaluate the relation between possible genetic abnormalities, pathological factors and prognosis. METHODS: Tumor tissue samples were obtained from 31 patients with NSCLC who underwent lobectomy with mediastinal lymph node dissection. Samples were analyzed by FISH using 8 alpha satellite DNA probe and c-myc gene cosmid probe. The relation between genetic abnormalities, pathological factors (T factor, tumor size, and N factor), and prognostic factors was evaluated by univariate and multivariate analysis, and by the Kaplan-Meier method and log-rank analysis. RESULTS: Chromosome 8 aberrations were T1 (n=3), 44.0%; T2 (n=18), 35.7%; T3 (n=7), 40.0%; T4 (n=3), 39.7% (p=NS). The c-myc gene amplifications were T1, 54.3%; T2, 51.1%; T3, 51.0%; T4, 66.3% (p=NS). There was no difference between patients whose tumor was more than 5 cm (n=16), and 5 cm or less (n=15) in the rate of chromosome 8 aberration (39.3%: 36.3%), or the rate of the c-myc gene amplification (52.1%: 53.7%). N factors for chromosome 8 aberrations were N0 (n=18), 35.9%; and N2 (n=11), 44.9% (p=NS). In the c-myc gene amplification, there was a significant difference between N0 and N2 (48.6%, 61.3%, p=0.040). In univariate and multivariate analysis, chromosome 8 aberrations correlated with a poor prognosis (p=0.037 and p=0.041). The 5-year survival rate was 15.4% in patients whose rate of chromosome 8 aberrations was 40% or more (n=13), which was significantly less than that in patients with an aberration rate of less than 40% (n=19, 57.9%, p=0.014). CONCLUSION: The c-myc gene amplification correlates with lymph node metastasis. Although there was no significant link between the amplification of the c-myc gene and clinical outcome, the numerical chromosome 8 aberrations was considered to be a factor for survival.

Two putative tumor suppressor genes on chromosome arm 8p may play different roles in prostate cancer.

Although loss of heterozygosity (LOH) on the short arm of chromosome 8 has been frequently observed in human prostate cancer, the relationship between LOH and clinical background is poorly understood. Fluorescence in situ hybridization (FISH) was employed to evaluate the chromosomal deletion on 8p in 42 prostate cancers using a centromeric probe for chromosome 8, in combination with 4 cosmid probes spanning 8p12 to 8p22. Deletions for at least one locus on the 8p were observed in 29 (69.0%) tumors. The most frequently deleted regions were 8p22 (54.8%) and 8p21.3 (52.4%), in almost the same frequency. The second most frequently deleted region was 8p21.1-p21.2 (38.1%). Deletions of 8p22 and 8p21.3 significantly correlated with tumor grade (P=0.0034, Fisher's exact probability test). A significantly higher frequency of the deletion on 8p21.1-p21.2 was observed in advanced prostate cancer (beyond capsular penetration or positive nodal metastases) than in localized prostate cancer (P=0.0033). In particular, deletion of 8p21.1-p21.2 was more frequently observed in the cases with lymph node metastases than without them (P=0.0029). No clinicopathological parameters had significant relation to deletions on 8p12. These results suggest that deletions on 8p22-p21.3 play an important role in tumor differentiation, while an 8p21.1-p21.2 deletion plays a role in the progression of prostate cancer.

Chromosomes exhibit preferential positioning in nuclei of quiescent human cells.

The relative spatial positioning of chromosomes 7, 8, 16, X and Y was examined in nuclei of quiescent (noncycling) diploid and triploid human fibroblasts using fluorescence in situ hybridization (FISH) with chromosome-specific DNA probes and digital imaging. In quiescent diploid cells, interhomolog distances and chromosome homolog position maps revealed a nonrandom, preferential topology for chromosomes 7, 8 and 16, whereas chromosome X approximated a more random distribution. Variations in the orientation of nuclei on the culture substratum tended to hinder detection of an ordered chromosome topology at interphase by biasing homolog position maps towards random distributions. Using two chromosome X homologs as reference points in triploid cells (karyotype = 69, XXY), the intranuclear location of chromosome Y was found to be predictable within remarkably narrow spatial limits. Dual-FISH with various combinations of chromosome-specific DNA probes and contrasting fluorochromes was used to identify adjacent chromosomes in mitotic rosettes and test whether they are similarly positioned in interphase nuclei. From among the combinations tested, chromosomes 8 and 11 were found to be closely apposed in most mitotic rosettes and interphase nuclei. Overall, results suggest the existence of an ordered interphase chromosome topology in quiescent human cells in which at least some chromosome homologs exhibit a preferred relative intranuclear location that may correspond to the observed spatial order of chromosomes in rosettes of mitotic cells.

Detection of AML1/ETO fusion transcript as a tool for diagnosing t(8;21) positive acute myelogenous leukemia.

The nonrandom chromosomal translocation t(8;21)(q22;q22) can be found frequently in acute myelogenous leukemia with maturation (AML-M2). The breakpoint of this translocation has been cloned and characterized, and fusion transcript AML1/ETO has been identified. Reverse transcription polymerase chain reaction (RT-PCR) can be used to amplify the breakpoint site of AML1/ETO in t(8;21)-positive AML-M2 patients. The chimeric transcript can be detected in all 16 (100%) t(8;21)-positive AML-M2 patients. In all samples, the size of the amplified DNA fragments and pattern of restriction digest were identical, indicating that the t(8;21) translocation breakpoint occurs within a single intron of the AML1 and ETO genes. Interestingly, this fusion transcript was also detected in one of 13 AML-M2 patients without the t(8;21) translocation, indicating that a masked translocation involving chromosomes 8 and 21, exists in AML. Minimal residual disease was detected by semi-nested RT-PCR in all four patients tested, who had been in complete remission for 12, 15, 34, and 52 months, respectively. These results indicate that RT-PCR amplification of the AML1/ETO fusion transcript is a powerful tool for diagnosing and monitoring minimal residual disease in AML-M2 patients.

Quantifying chromosome changes and lineage involvement in myelodysplastic syndrome (MDS) using fluorescent in situ hybridization (FISH).

A simplified technique for fluorescent in situ hybridization (FISH) was used to investigate the prevalence of chromosomally abnormal clones in 13 cases of myelodysplastic syndrome (MDS). Biotinylated centromeric probes for chromosomes 7, 8, 12 and X, as well as painting probes for chromosomes 7 and 11, were applied to air-dried bone marrow smears stored from 6 to 23 months. Nine of the cases had been previously karyotyped, and five of these demonstrated normal karyotypes which were confirmed by FISH. The remaining four cases showed different chromosome changes. One case of sideroblastic anemia with chronic lymphocytic leukemia showed minor clones with either monosomy 12 (12% of cells) or tetraploidy (15% of cells) by FISH, whereas metaphase cytogenetics had demonstrated trisomy 12 in 20% of cells, with no evidence of tetraploidy. Another case which had been previously karyotyped was found to have a t(7;11) in 90% of cells while only 10% of cells were shown by FISH to contain this translocation. Monosomy 7 was demonstrated by FISH in a case of refractory anemia (RA), while trisomy 8 was found in a case of RA with excess blasts in transformation (RAEB-T), and in both of these cases the aneuploid clone was present in eosinophils as well as in erythroid and granulocytic precursors but not in lymphocytes or histiocytes, thereby demonstrating the value of FISH for identifying the affected cell lineage.

Human chromosome 8 (p12-->q22) complements radiosensitivity in the severe combined immune deficiency (SCID) mouse.

The severe combined immune deficiency (SCID) mouse shows two kinds of phenotypic abnormalities, a high radiosensitivity and an abnormal immunoglobulin gene recombination. A genetic study has revealed that a mutation exists in chromosome 16. However, several attempts to isolate the gene responsible for these phenotypes have been unsuccessful. By making use of the characteristics of radiosensitivity, we conducted complementation experiments to identify a human chromosome which contains the responsible gene. Radioresistant cells were selected from the hybrid cells of the SCID mouse and human fibroblasts. Based on this approach, the gene complementing the SCID phenotype was assigned to human chromosome 8 p12-->q22.

Acute myeloblastic leukemia (ANLL-M2) with t(8;21)(q22;q22) variant expressing lymphoid but not myeloid surface antigens with a high number of G-CSF receptors.

Leukemic cells from an 8-year-old girl with ANLL-M2 expressed precursor B-cell antigen CD19, but none of the myeloid antigens CD11b, CD13, CD14 and CD33. After culture, the cells expressed CD11b and CD13. The cells carried a high number of granulocyte colony-stimulating factor (G-CSF) receptors. In chromosome analysis, metaphase cells were obtained only in the case of culture with G-CSF. The karyotype was a variant of t(8;21)(q22;q22). Southern blot analysis revealed rearrangement of the AMLI gene located on chromosome 21. These observations may suggest that even without myeloid surface antigens and with precursor B-cell antigen, ANLL-M2 with t(8;21)(q22;q22) has apparent myeloid characteristics.

Chromosomes 8, 12, and 17 copy number in Astler-Coller stage C colon cancer in relation to proliferative activity and DNA ploidy.

Fluorescence in situ hybridization using centromere-specific DNA probes to chromosomes 8, 12, and 17 was applied to 23 archival paraffin-embedded stage C colonic cancer specimens. Chromosome copy number was related to flow cytometric determinations of S-phase fraction and DNA ploidy. Three to eight copies of chromosomes 8, 12, and 17 were observed at mean frequencies of 28.7%, 37.8%, and 20.9%, respectively. The mean frequency of multiple copies of chromosome 12 was significantly greater than that for chromosome 17 (P < 0.0025). The mean frequency of single copies of chromosome 17 was significantly greater than that for chromosomes 8 and 12 (P < 0.0025 and P < 0.0005, respectively). Regarding the fourth quartile of cases, defined on the basis of the frequency of multiple chromosome copies, the proportion demonstrating moderate to high proliferative activity greatly exceeded the proportion displaying low proliferative activity. The same cases (most chromosomally aberrant) also generally demonstrated DNA aneuploidy. The results indicate a substantial degree of karyotypic instability in advanced colon cancer, particularly in cases with high proliferative activity and DNA aneuploidy.

Atypical chronic myelogenous leukemia in a patient with trisomy 8 mosaicism syndrome.

A 17-year-old woman was admitted for bone marrow transplantation with the diagnosis of atypical Philadelphia-negative chronic myelogenous leukemia (aCML), cytogenetically characterized by trisomy 8 as the sole chromosome aberration. A striking feature was a congenital opacity of the right cornea. Chromosomal analysis of skin fibroblasts were performed and revealed a mosaic for trisomy 8. Commonly, a distinct clinical picture leads to the diagnosis of trisomy 8 mosaicism syndrome (T8ms), but an extreme phenotypic variability has been observed. To our knowledge the development of an aCML in a patient with T8ms has not been reported. A review of the literature revealed that an association to other hematological disorders had been described in two cases. The question of whether our patient's aCML was a random event or not is discussed. The patient is now 24 months post transplant and shows no evidence of disease. Her Karnofsky score is 100%. We conclude that it might be worthwhile to look for an associated constitutional trisomy 8 mosaicism in all patients with trisomy 8 leukemia.

Frequent loss of heterozygosity for loci on chromosome 8p in hepatocellular carcinoma, colorectal cancer, and lung cancer.

Frequent loss of heterozygosity at chromosomal loci in a specific tumor type may indicate the presence of a tumor suppressor gene. We have examined loss of heterozygosity on chromosome 8p in paired tumor and constitutional DNA from 346 patients representing seven different types of human cancer. Frequent allelic losses were observed in hepatocellular carcinoma (22 of 46 cases, 47.8%), in colorectal cancer (12 of 26, 46.2%), and in non-small cell lung cancer (14 of 35, 40.0%), in contrast to low frequencies detected in breast cancer (5 of 56, 8.9%) and renal cell carcinoma (2 of 27, 7.4%). Ovarian cancer and gastric cancer showed intermediate frequencies of 33.3% and 22.2%. Subsequent analysis of 120 hepatocellular carcinomas and 94 colorectal cancers with five polymorphic markers along the short arm of chromosome 8 defined commonly deleted regions within the same chromosomal interval, 8p23. 1-8p21.3, suggesting that one or more tumor suppressor genes for both cancers may be present in that region.

Molecular cytogenetic analysis of a familial 8p23.1 deletion associated with minimal dysmorphic features, seizures, and mild mental retardation.

We report a family in which three members presented with minimal phenotypic abnormalities, normal intelligence to mild mental retardation, and a cytogenetically terminal chromosome deletion at band 8p23.1 Whole chromosomal painting with a chromosome 8-specific DNA library confirmed this familial chromosome abnormality as a deletion, while fluorescence in situ hybridization with telomeric probes demonstrated the presence of telomeres at the deletion site. Coagulation studies were additionally performed to evaluate the purported location of the coagulation factor VII regulator gene at 8p23.1. A review of the clinical findings of seven cases of del(8)(p23.1) is presented.

Evidence for a new tumor-suppressor gene involved in gastrointestinal malignancies.

Inactivation or loss of tumor-suppressor genes is believed to lead to the development or progression of malignancies. To determine whether a tumor-suppressor gene is located on chromosome 8, DNA was extracted from tumor and normal tissue of colorectal, gastric, and pancreatic specimens, and allele loss was investigated by Southern hybridization techniques with the chromosome 8 probe D8S7. Twenty-five percent of pancreatic carcinomas, 50% of gastric carcinomas, and 50% of colorectal carcinomas were found to have lost an allele on chromosome 8. These findings suggest the presence of a tumor-suppressor gene on chromosome 8, which is involved in colorectal carcinoma, gastric carcinoma, and pancreatic carcinoma. Definition of the frequency with which this tumor-suppressor gene is involved in gastrointestinal malignancies will await the study of many patients who are classified as informative and the use of multiple probes for chromosome 8.