Hypermethylation and loss of retinoic acid receptor responder 1 expression in human choriocarcinoma.

BACKGROUND: Human placental development resembles tumorigenesis, due to the invasive and fusogenic potential of trophoblasts. However, these features are tightly controlled in trophoblasts. Disturbance of this spatial and temporal regulation is thought to contribute to the rare formation of choriocarcinomas. Promoter hypermethylation and loss of the tumor suppressor Retinoic acid receptor responder 1 (RARRES1) were shown to contribute to cancer progression. Our study investigated the epigenetic and transcriptional regulation of RARRES1 in healthy human placenta in comparison to choriocarcinoma cell lines and cases. METHODS: Three choriocarcinoma cell lines (Jeg-3, JAR and BeWo) were treated with three different retinoic acid derivates (Am580, Tazarotene and all-trans retinoic acid) and 5-aza-2'-deoxycytidine. We analyzed RARRES1 promoter methylation by pyrosequencing and performed realtime-PCR quantification to determine RARRES1 expression in placental tissue and trophoblastic cell lines. Additionally, RARRES1 was stained in healthy placentas and in biopsies of choriocarcinoma cases (n = 10) as well as the first trimester trophoblast cell line Swan71 by immunofluorescence and immunohistochemistry. RESULTS: In the choriocarcinoma cell lines, RARRES1 expression could not be induced by sole retinoic acid treatment. Stimulation with 5-aza-2'-deoxycytidine significantly induced RARRES1 expression, which then could be further increased with Am580, Tazarotene and all-trans retinoic acid. In comparison to healthy placenta, choriocarcinoma cell lines showed a hypermethylation of the RARRES1 promoter, which correlated with a reduced RARRES1 expression. In concordance, RARRES1 protein expression was lost in choriocarcinoma tissue. Additionally, in the trophoblastic cell line Swan71, we found a significant induction of RARRES1 expression with increased cell density, during mitosis and in syncytial knots. CONCLUSIONS: Our findings showed that RARRES1 expression is absent in choriocarcinoma due to promoter methylation. Based on our analysis, we hypothesize that RARRES1 might exert tumor suppressive functions in multiple cellular processes (e.g. cell cycle regulation, adhesion, invasion and apoptosis).

Contribution of different placental cells to the expression and stimulation of antimicrobial proteins (AMPs).

The placenta is a major barrier that prevents potentially infectious agents from causing fetal diseases or related complications during pregnancy. Therefore, we postulated that the placenta might express a broad repertoire of antimicrobial proteins as well as inflammatory chemokines and cytokines to combat invading microorganisms. Here we demonstrate that placental cells indeed express a wide range of AMPs (antimicrobial peptides and proteins) including bactericidal/permeability-increasing protein (BPI), secretory leukocyte protease inhibitor (SLPI), human ß-defensin 2 (hBD2), acyloxyacyl hydrolase (AOAH), and cathelicidin (CAP18). In addition, these cells also secrete pro-inflammatory cytokines and chemokines upon stimulation with bacterial ligands. Notably, we show that BPI expression by placental cells could be completely attributed to granulocytes while highly purified placental trophoblasts expressed only a subset of the AMPs like SLPI. Unexpectedly, trophoblast AMPs did not exhibit inducible secretion in response to various TLR ligands and further investigations showed that the unresponsiveness of trophoblasts to lipopolysaccharide (LPS) was due to a lack of TLR4 expression. In summary, we have shown that the expression of different AMPs can be allocated to various cells in the placenta and the repertoire of the AMPs expressed by placental cells is a result of a cooperation of leukocytes as well as cells from embryonic origin.

Risk Factors for Endometriosis in a German Case-Control Study.

Objective: The etiology of endometriosis is still a research field in which few consistent data are available. Large case-control studies or even cohort studies are rare, and most of the published data are conflicting. The aim of the present study was therefore to examine common epidemiological and endometriosis-specific risk factors in a German case-control study. Design: From 2001 to 2010, a pool of 595 laparoscopically confirmed cases and 475 controls were recruited in a hospital-based setting. After matching for age, 298 cases and 300 controls remained in the pool. Age at menarche, menstrual cycle length, duration of menstrual bleeding, number of pregnancies, live births, miscarriages, use of contraceptive pills, body mass index (BMI), and smoking status were analyzed with logistic regression models predicting endometriosis case-control status. Results: Menstrual cycle length, duration of menstrual bleeding, number of pregnancies, number of miscarriages, and smoking status, as relevant predictors for endometriosis case-control status, were identified as risk factors for endometriosis. Other factors such as age at menarche, number of live births, ever having used contraceptive pills, and BMI were not predictive. Conclusions: This hospital-based case-control study reproduced most of the familiar risk factors. Comparison of this study with others reveals a wide variety of effect sizes and directions of association with risk factors and may increase the information available about the characteristics of the patient population being treated in the relevant hospital setting.

Regulation of the NRSF/REST gene by methylation and CREB affects the cellular phenotype of small-cell lung cancer.

The neuron-restrictive silencer factor/RE1-silencing transcription factor (NRSF/REST) is a negative regulator of gene expression restricting the expression of neuronal genes to the nervous system. NRSF/REST is highly expressed in non-neuronal tissues like the lung. In previous work, we identified small-cell lung cancer (SCLC) cell lines with no detectable NRSF/REST expression that, as a consequence, expressed neuronal markers like L1-cell adhesion molecule (L1-CAM) and neural cell adhesion molecule (NCAM). The loss of NRSF/REST expression was linked to malignant progression; however, its mechanistic role remained elusive. Here, we show that NRSF/REST itself, rather than one of its regulated genes, acts like a classic tumour suppressor, being in part regulated by methylation. In SCLCs, NRSF/REST is positively regulated by CREB, with an NRSF/REST promoter fragment showing cell type specificity. Downstream, NRSF/REST directly regulates AKT2, in which NRSF/REST loss leads to an epidermal growth factor-mediated de-regulation of AKT-Serine473 phosphorylation, important for cellular proliferation and survival. Assaying anchorage-independent growth, we observed that with reduced NRSF/REST expression, proliferation was significantly enhanced, whereas NRSF/REST rescue decreased the potential of cells to grow anchorage independently. Our observations support the fact that NRSF/REST may act as an important modulator of malignant progression in SCLC.

Polymorphisms in estrogen metabolism and estrogen pathway genes and the risk of miscarriage.

PURPOSE: This study investigated genetic variations in the estrogen pathway and their association with miscarriages. METHODS: A total of 483 patients were recruited from a comprehensive control group for case-control studies. Three variants of the CYP19A1 gene (rs10046, rs4646 and rs700519) and one variant each of the estrogen (ESR1) and progesterone (PGR) receptor genes (rs3020314 and rs1042838) were investigated using polymorphism genotyping. The chi-squared test and one-way analysis of variation (ANOVA) were used for statistical analysis. RESULTS: For rs10046 (CYP19A1), the C/C genotype was associated with a greater frequency of miscarriages (P = 0.017). The other genotypes were not found to be associated with recurrent miscarriage. CONCLUSIONS: This is the first study that has identified a single-nucleotide polymorphism in the aromatase gene that suggests a significant association between genotypes and miscarriage. As aromatase is an essential enzyme in the estrogen pathway, it may be speculated that variations in the aromatase gene in some way give rise to different conditions in the endocrine environment that can lead to impaired fertility.

Assessment of pituitary and steroid hormones and members of the TGF-beta superfamily for ovarian function in patients with congenital uterus and vaginal aplasia (MRKH syndrome).

Patients with Mayer-Rokitanski-Kuster-Hauser (MRKH) syndrome have congenital uterine and vaginal aplasia. The main question of this study was, if the absence of a uterus along with other genital and organ malformations could contribute to hormone or other growth factor protein fluctuations involved in communication between the hypothalamus-pituitary axis, ovaries and uterus. Serum from 56 MRKH patients (mean 27.6 years) and 22 female controls (mean 30.7 years) were analyzed using ELISA to determine levels of pituitary and steroid hormones (LH, FSH, estradiol, progesterone), growth factors of the TGF-beta superfamily like activin A, inhibin B, and anti-Müllerian hormone (AMH). All serum levels were analyzed in relation to other organ malformations. Compared to controls, all 56 patients, including 5% with streak ovaries or unilateral ovarian aplasia, were generally similar in hormone and growth factor levels and could be grouped into hormonal phases. However, compared to controls LH/FSH and FSH/LH ratios of patients had significantly higher and lower mean values, of 2.75-fold (p=0.015) and 1.9-fold (p=0.002), respectively. Undetectable inhibin B levels of<10 pg/ml (p=0.05) were noted in 41.1% of MRKH patients, resulting in significantly higher activin A/inhibin B ratios (p<0.001). MRKH patients have hormonal phases supporting ovarian function, but patients with low FSH/LH ratios and undetectable inhibin B levels (<10 pg/ml) could represent cycle phasing irregularities. A model is discussed regarding our findings and the loss of ovarian-uterine communication.

Polymorphisms in the novel serotonin receptor subunit gene HTR3C show different risks for acute chemotherapy-induced vomiting after anthracycline chemotherapy.

The aim of this study was to correlate chemotherapy-induced nausea and vomiting (CINV) with commonly occurring single nucleotide polymorphisms (SNP) in the 5-hydroxytryptamine receptor 3 genes (HTR3). Women with breast cancer without previous chemotherapy were eligible for this prospective study. All patients received epirubicin, with or without cyclophosphamide, and preventive medication with ondansetron and dexamethasone. The patients documented every vomiting event on an hourly basis. Real-time polymerase chain reaction (PCR) analysis was performed for the following nonsynonymous SNPs: p.Y129S (HTR3B), p.K163N (HTR3C) and p.A405G (HTR3C). The overall proportion of patients (total n = 110) who reported vomiting in the first 24 h after chemotherapy was 31.8%. The variant genotype of K163N (HTR3C) was associated with vomiting, which occurred in 50.0% (P = 0.009). Polymorphisms in the HTR3C gene could serve as a predictive factor for CINV in patients undergoing moderately emetogenic chemotherapy.

Higher incidence of linked malformations in siblings of Mayer-Rokitansky-Küster-Hauser-syndrome patients.

BACKGROUND: Mayer-Rokitansky-Küster-Hauser (MRKH) syndrome is a malformation of the female genital tract (vaginal aplasia, rudimentary uterus, normal fallopian tubes and high ovaries). The incidence is one in 4000 female newborns. The aim of the present study was to record genital and associated malformations among siblings and relatives of MRKH patients in order to draw possible conclusions regarding the etiology of the syndrome: heredity (dominant versus recessive) or spontaneous malformation. METHODS: Using a standardized questionnaire, affected MRKH patients were asked about other cases of MRKH and/or associated malformations among siblings and relatives. RESULTS: No other cases of MRKH syndrome had occurred among the siblings or relatives of 73 MRKH patients; however, 13 associated malformations were recorded among a total of 103 siblings. Musculoskeletal malformations were markedly increased (3.27 times higher) in comparison with the prevalence of congenital malformations among newborns in the normal population. CONCLUSIONS: This study shows that dominant inheritance cannot play a role in the etiology of MRKH syndrome, as no further cases of MRKH syndrome occurred among any of the siblings. The study provides support for the view that the syndrome has a multifactorial pathogenesis. Siblings/relatives of MRKH patients should be examined for associated musculoskeletal/urogenital malformations.

Single nucleotide polymorphism D1853N of the ATM gene may alter the risk for breast cancer.

PURPOSE: Various ATM (ataxia telangiectasia-mutated) mutations and polymorphisms have been reported to be associated with an increased breast cancer risk. Recent studies have produced contradictory results regarding the association between ATM genetic variants and breast cancer risk. MATERIALS AND METHODS: The common ATM polymorphism 5557G>A (p.D1853N) (rs1801516), previously suggested to be associated with bilateral breast cancer, was analyzed using real-time PCR in 514 unselected patients with breast cancer and 511 age-matched healthy control individuals. DNA was obtained from peripheral blood draw. RESULTS: The ATM genotype was weakly associated with the risk for breast cancer (P = 0.04 for the overall test). The odds ratio for women with a heterozygous genotype was 0.70 (95% CI, 0.52-0.94) and for the homozygous variant 0.63 (95% CI, 0.27-1.49). Disease-free survival and overall survival showed no significant association with specific genotypes. CONCLUSIONS: The results of this study might suggest a minor association between polymorphism 5557G>A and a reduced risk of breast cancer.

Evaluation of clinical parameters and estrogen receptor alpha gene polymorphisms for patients with endometriosis.

Endometriosis is a chronic inflammatory disease, which is especially found in women with subfertility problems with an incidence of up to 30%. The disease is considered an estrogen-dependent disorder, where DNA polymorphisms of the estrogen receptor alpha (ERalpha) in connection with endometriosis are controversially discussed. From a German population of women, clinical data associated with the disease, including the American Fertility Society (AFS) I-IV classification, and non-clinical parameters were evaluated statistically in endometriosis patients (n = 98) and in control women (n = 98) without endometriosis. Using a multivariate statistical analysis, significant associations of endometriosis with dysmenorrhea (P < 0.001) and allergies against medicaments (P = 0.042) were found. A positive trend between first grade family history of endometriosis and allergies against medicaments was also observed, suggesting a genetic relationship. From both collectives, DNA from peripheral blood was analyzed for the frequency of the ERalpha DNA polymorphisms Xba1 (A/G) and PvuII (T/C) in intron 1 and the ERalpha exonic DNA polymorphism (G229A) with an amino acid exchange (Gly77Ser) in the transactivation domain. DNA samples from endometriosis lesions and control tissues from the same collectives were also analyzed for the exonic G229A polymorphism. Only homozygote wild-type alleles for the polymorphism G229A were found, making it a rare polymorphism in mid-European individuals. Allele types for the PvuII and Xba1 polymorphisms were analyzed with the observed statistically significant clinical parameters and showed no significant association with endometriosis; however a trend with AFS IV was noted, which could contribute to lesion severity. In conclusion, the analyzed polymorphisms in the ERalpha do not have a functional role concerning specific clinical parameters associated with endometriosis.

DNA sequence variations of the entire anti-Mullerian hormone (AMH) gene promoter and AMH protein expression in patients with the Mayer-Rokitanski-Kuster-Hauser syndrome.

BACKGROUND: The etiology of the Mayer-Rokitanski-Kuster-Hauser (MRKH) syndrome, where congenitally the Mullerian ducts fail to develop into the uterus, cervix and upper vagina, along with other malformations, is unresolved. Anti-Mullerian hormone (AMH) signal transduction inducing the degradation of Mullerian ducts in males is implicated in the MRKH syndrome. This study examined if DNA sequence variations are responsible for the activation of AMH and aberrant hormone levels in MRKH patients. METHODS: The entire AMH promoter and 3' regulatory elements of the constitutively expressed splicing factor SF3a2 were sequenced in 30 MRKH patients and genotyped in 48 control individuals using matrix-assisted laser desorption/ionization-time-of-flight mass spectronomy. Ovarian AMH promoter function was correlated with protein expression in plasma and peritoneal fluid of MRKH patients. RESULTS: Of six identified AMH promoter variations, two at positions -639 (SP1-binding site) and -210 [steroidogenic factor (SF)1-binding site] were homozygote in 73% of patients, and 69% of control individuals, destroying the SP1-binding site. AMH protein levels in plasma and peritoneal fluid from patients were equivalent to control individuals, however in three patients plasma levels were abnormally high. CONCLUSIONS: AMH is an important indicator for ovarian function. AMH promoter sequence variations or the previously proposed SF3a2-AMH fusion co-transcripts cannot be responsible for aberrant AMH expression leading to Mullerian duct degradation.

Cation-chromatin binding as shown by ion microscopy is essential for the structural integrity of chromosomes.

Mammalian interphase and mitotic cells were analyzed for their cation composition using a three-dimensional high resolution scanning ion microprobe. This instrument maps the distribution of bound and unbound cations by secondary ion mass spectrometry (SIMS). SIMS analysis of cryofractured interphase and mitotic cells revealed a cell cycle dynamics of Ca2+, Mg2+, Na+, and K+. Direct analytical images showed that all four, but no other cations, were detected on mitotic chromosomes. SIMS measurements of the total cation content for diploid chromosomes imply that one Ca2+ binds to every 12.5-20 nucleotides and one Mg2+ to every 20-30 nucleotides. Only Ca2+ was enriched at the chromosomal DNA axis and colocalized with topoisomerase IIalpha (Topo II) and scaffold protein II (ScII). Cells depleted of Ca2+ and Mg2+ showed partially decondensed chromosomes and a loss of Topo II and ScII, but not hCAP-C and histones. The Ca2+-induced inhibition of Topo II catalytic activity and direct binding of Ca2+ to Topo II by a fluorescent filter-binding assay supports a regulatory role of Ca2+ during mitosis in promoting solely the structural function of Topo II. Our study directly implicates Ca2+, Mg2+, Na+, and K+ in higher order chromosome structure through electrostatic neutralization and a functional interaction with nonhistone proteins.

DNA structural properties of AF9 are similar to MLL and could act as recombination hot spots resulting in MLL/AF9 translocations and leukemogenesis.

The human AF9 gene at 9p22 is one of the most common fusion partner genes with the MLL gene at 11q23, resulting in the t(9;11)(p22;q23). The MLL-AF9 fusion gene is associated with de novo acute myelo-genous leukemia (AML), rarely with acute lymphocytic leukemia (ALL) and with therapy related leukemia (t-AML). The AF9 gene is >100 kb and two patient breakpoint cluster regions (BCRs) have been identified; BCR1 is within intron 4, previously called site A, whereas BCR2 or site B spans introns 7 and 8. Patient breakpoint locations were determined previously by RT-PCR and by genomic DNA cloning. In this study, we defined the exon-intron boundaries and identified several different structural elements in AF9 including a co-localizing in vivo DNA topo II cleavage site and an in vitro DNase I hypersensitive (DNase 1 HS) site in intron 7 in BCR2. Reversibility experiments demonstrated a religation of the topo II cleavage sites. The location of the in vivo topo II cleavage site was confirmed in vitro using a topo II cleavage assay. In addition, two scaffold associated regions (SARs) are located centromeric to the topo II and DNase I HS cleavage sites and border both patient breakpoint regions: SAR1 is located in intron 4, whereas SAR2 encompasses parts of exons 5-7. This study demonstrates that the patient breakpoint regions of AF9 share the same structural elements as the MLL BCR. We describe a DNA breakage and repair model for non-homologous recombination between MLL and its partner genes, particularly AF9.

Dietary bioflavonoids induce cleavage in the MLL gene and may contribute to infant leukemia.

Chromosomal translocations involving the MLL gene occur in about 80% of infant leukemia. In the search for possible agents inducing infant leukemia, we identified bioflavonoids, natural substances in food as well as in dietary supplements, that cause site-specific DNA cleavage in the MLL breakpoint cluster region (BCR) in vivo. The MLL BCR DNA cleavage was shown in primary progenitor hematopoietic cells from healthy newborns and adults as well as in cell lines; it colocalized with the MLL BCR cleavage site induced by chemotherapeutic agents, such as etoposide (VP16) and doxorubicin (Dox). Both in vivo and additional in vitro experiments demonstrated topoisomerase II (topo II) as the target of bioflavonoids similar to VP16 and Dox. Based on 20 bioflavonoids tested, we identified a common structure essential for topo II-induced DNA cleavage. Reversibility experiments demonstrated a religation of the bioflavonoid as well as the VP16-induced MLL cleavage site. Our observations support a two-stage model of cellular processing of topo II inhibitors: The first and reversible stage of topo II-induced DNA cleavage results in DNA repair, but also rarely in chromosome translocations; whereas the second, nonreversible stage leads to cell death because of an accumulation of DNA damage. These results suggest that maternal ingestion of bioflavonoids may induce MLL breaks and potentially translocations in utero leading to infant and early childhood leukemia.

An in vivo topoisomerase II cleavage site and a DNase I hypersensitive site colocalize near exon 9 in the MLL breakpoint cluster region.

The human myeloid-lymphoid leukemia gene, MLL (also called ALL-1, Htrx, or HRX ), maps to chromosomal band 11q23. MLL is involved in translocations that result in de novo acute lymphoblastic leukemia (ALL), acute myelogenous leukemia (AML), mixed lineage leukemia, and also in therapy AML (t-AML) and therapy ALL (t-ALL) resulting from treatment with DNA topoisomerase II (topo II) targeting drugs. MLL can recombine with more than 30 other chromosomal bands, of which 16 of the partner genes have been cloned. Breaks in MLL occur in an 8. 3-kb breakpoint cluster region (BCR) encompassing exons 5 through 11. We recently demonstrated that 75% of de novo patient breakpoints in MLL mapped in the centromeric half of the BCR between two scaffold-associated regions (SAR), whereas 75% of the t-AML patient breakpoints mapped to the telomeric half of the BCR within a strong SAR. We have mapped additional structural elements in the BCR. An in vivo DNA topo II cleavage site (induced with several different drugs that target topo II) mapped near exon 9 in three leukemia cell lines. A strong DNase I hypersensitive site (HS) also mapped near exon 9 in four leukemia cell lines, including two in which MLL was rearranged [a t(6;11) and a t(9;11)], and in two lymphoblastoid cell lines with normal MLL. Two of the leukemia cell lines also showed an in vivo topo II cleavage site. Our results suggest that the chromatin structure of the MLL BCR may influence the location of DNA breaks in both de novo and therapy-related leukemias. We propose that topo II is enriched in the MLL telomeric SAR and that it cleaves the DNase I HS site after treatment with topo II inhibitors. These events may be involved in recombination associated with t-AML/t-ALL breakpoints mapping in the MLL SAR.

Scaffold-associated regions in the human type I interferon gene cluster on the short arm of chromosome 9.

Scaffold-associated regions (SARs) function at the level of modeling or shaping the chromatin of DNA into loop domains. We have mapped 36 SARs in the human type I interferon (IFN) gene complex on chromosome 9, band p21-22, to examine the overall structure of this gene complex. A total of 29 strong SARs and 7 weak SARs were mapped to the flanking regions of the different interferon genes. Twenty-two strong SARs mapped to the flanking regions of 13 interferon (IFNA) alpha genes; 2 strong SARs mapped to one interferon omega (IFNW) gene; 2 strong SARs mapped to one interferon alpha pseudogene (IFNAP); and 3 strong SARs mapped to two interferon omega pseudogenes (IFNWP). One weak SAR mapped to the flanking region of one IFNA gene, whereas 6 weak SARs flanked four IFN pseudogenes (P11, P12 P20, P23). The IFN SAR structure was comparable between the BV173 leukemia cell line and the U373 glioma cell line. Analysis of two glioma deletion breakpoint junctions, where breaks occur within and outside the IFN gene cluster, revealed an association with SARs. IFN SARs showed evidence for cooperativity among the SARs, while DNA sequence analysis revealed a series of clustered A-tracts within strong SARs. These data suggest that the IFN genes may be organized into a series of small (2-10 kb) DNA loop domains, with each loop containing a coding region flanked by SARs. In our model, the SAR enrichment and the clustering of A-tracts observed at the SARs within the IFN gene complex represent a higher level of chromatin organization, which may predispose this region to breakage.

Identification of complex genomic breakpoint junctions in the t(9;11) MLL-AF9 fusion gene in acute leukemia.

The MLL gene at chromosome 11, band q23, is involved in translocations with as many as 40 different chromosomal bands. Virtually all breakpoints occur within an 8.3 kb BamHI fragment and result in 5' MLL fused to partner genes in a 5'-3' orientation. The translocation t(9;11)(p22;q23), which results in the fusion of MLL to AF9, is the most common of the 11q23 chromosomal abnormalities observed in de novo acute myeloid leukemia (AML), in therapy related leukemia (t-AML), and rarely in acute lymphoblastic leukemia (ALL). We have studied 24 patients with a t(9;11) and an MLL rearrangement, including 19 patients with AML, four with t-AML, and one with ALL. To understand the mechanisms of this illegitimate recombination, we cloned and sequenced the t(9;11) translocation breakpoint junctions on both derivative chromosomes from one AML patient and from the Mono Mac 6 (MM6) cell line, which was derived from a patient with AML. Two different complex junctions were noted. In the AML patient, both chromosome 11 and 9 breaks were staggered, occurred in Alu DNA sequences, and resulted in a 331 bp duplication. In the MM6 cell line, breaks in chromosomes 11 and 9 were also staggered, but, in contrast to the finding in the AML patient, the breaks did not involve Alu DNA sequences and resulted in a 664 bp deletion at the breakpoints. Using reverse transcriptase (RT-) PCR, we analyzed 11 patient samples, including the two just described, for MML-AF9 fusions. The fusion occurred in six of seven AML patients, two of two t-AML patients, one patient with ALL, and in the MM6 cell line. Interestingly, all of the breaks within the AF9 gene in AML patients occurred in the central AF9 exon, called Site A by others, whereas in the single ALL patient the breakpoint mapped to a more 3' region of the AF9 gene. Our data, when combined with those of others, suggest that the fusion point within the AF9 gene, and thus the amount of AF9 material included in the MLL-AF9 fusion gene product, may influence the phenotype of the resulting leukemia. This further supports the proposal that the MML translocation partner genes play a critical role in the leukemogenic process.

AF10 is split by MLL and HEAB, a human homolog to a putative Caenorhabditis elegans ATP/GTP-binding protein in an invins(10;11)(p12;q23q12).

Invins(10;11)(p12;q23q12) is one of the rare but recurring chromosome rearrangements seen in acute monoblastic leukemia. We cloned the proximal 10p breakpoint from one patient and showed that the MLL gene at 11q23 was fused to the 3' portion of AF10 at 10p12. In addition, we cloned the telomeric 10p junction and we found that the 5' portion of AF10 was juxtaposed to a previously unidentified gene at 11q12, which we call HEAB (a human homolog to a hypothetical Caenorhabditis elegans ATP/GTP-binding protein). These results indicate that the AF10 gene is split into a 5' AF10 and a 3' AF10 portion by the 11q23q12 chromosome segment and that both breakpoint junctions result in fusion transcripts of 5' AF10/HEAB and MLL/3' AF10. Only the MLL/3' AF10 fusion mRNA results in an in-frame fusion. Northern blot analysis of HEAB expression shows that a 2.0-kb major transcript is expressed ubiquitously in human tissues and is especially abundant in testis and skeletal muscle, whereas a 3.2-kb minor transcript is noted with the highest level of expression in thymus and peripheral blood leukocytes. The HEAB gene encodes a 425-amino acid protein that is rich in valine and leucine. HEAB protein shows high homology in its entire amino acid sequence to a putative C elegans protein and contains an adenosine triphosphate (ATP)/guanosine triphosphate (GTP)-binding motif that has homology to the ATP-binding transporter superfamily or to GTP-binding proteins. Our results could explain the high frequency of complex insertion and other rearrangement events that involve 10p12 and 11q12 and 11q23. The finding that different portions of a single gene are involved in fusions with two independent genes in the same leukemic cell is unique in the analysis of chromosome translocations.

Scaffold attachment regions in centromere-associated DNA.

Due to indications that kinetochore proteins are an integral part of the protein scaffold component of the chromosome (Earnshaw et al. 1984), we chose to map the distribution of scaffold attachment regions (SARs) at centromeres. Using the SAR mapping assay of Mirkovitch et al., Southern blots were prepared and probed with 32P-labeled fragments from the human 1.9 kb centromeric alpha-satellite repeat unit of chromosome 1 or the 1.7 kb centromeric alpha-satellite repeat unit of chromosome 16. Our results demonstrated the presence of one SAR site per 1.9 kb repeat unit in chromosome 1, and every 1.7 kb repeat unit in chromosome 16, separated by regions of small DNA loops over the length of the alpha-satellite regions. We also identified several in vitro vertebrate topoisomerase II and cenP-B consensus sequences throughout the chromosome 1 alpha-satellite region using computer and base ratio analysis, to address the question as to why some alpha-satellite regions are SAR related and others are not. To provide in situ indications of SAR localization in the human genome, SAR DNA and non-SAR DNA were prepared following lithium 3,5-di-iodosalicylate extraction. Sequences protected from DNAse I digestion by SAR proteins, as compared with unprotected DNA that was digested by the enzyme, was labeled with biotin-UTP, hybridized to chromosomal DNA in situ, and then detected with fluorescein-avidin-DCS. Both SAR and non-SAR DNA selectively labeled virtually all centromeric regions of the human metaphase karyotype. Chromosomal arms were less strongly bound by SAR DNA, with a pattern that followed the chromosomal axis. In the more condensed chromosomes an R-banding pattern was evident. In general, labeling patterns produced by both SAR and non-SAR fractions were similar, as expected from the indications that SAR DNAs are heterogenous in sequence and do not form a specific class of sequences. We conclude that centromeric regions of several, possibly all, human metaphase chromosomes are also regions where the chromosomal axis contains loops, smaller in size than in the arms and where attachment sites are concentrated. This clustering of SARs may be responsible in part for the tight chromatin packing associated with the primary constriction of the centromeric region.