Association of methylenetetrahydrofolate reductase polymorphism C677T and dietary folate with the risk of cervical dysplasia.

Epidemiological studies have been inconsistent regarding a role for folate in the etiology of cervical dysplasia. Methylenetetrahydrofolate reductase (MTHFR) catalyzes the synthesis of 5-methyltetrahydrofolate, which is involved in the methylation of homocysteine to methionine. A common variant of this enzyme, resulting from a 677C-->T (Ala-->Val) substitution in the gene, has been shown to have reduced activity and is associated with mild hyperhomocysteinemia. A multiethnic case-control study was used to examine the association of dietary folate and MTHFR genotype with the odds ratios (ORs) for cervical dysplasia among women identified from several clinics on Oahu, Hawaii, between 1992 and 1996. We collected blood samples for DNA extraction, cervical smears for cytological diagnosis, exfoliated cervical cells for human papillomavirus (HPV) DNA testing, and personal interviews from 150 women with squamous intraepithelial lesions (SILs) and from 179 women with cytologically normal (Pap) smears. We found a positive, monotonic trend (P = 0.02) in the ORs for cervical SILs associated with the number of variant MTHFR T alleles, after multivariate adjustment. Women with the heterozygous CT genotype had twice the risk of cervical SILs [OR, 2.0; 95% confidence interval (CI), 1.1-3.7], and women with the homozygous TT genotype had almost three times the risk of SILs (OR, 2.9; 95% CI, 1.0-8.8) compared to women with the homozygous MTHFR CC genotype. The dietary intakes of folate, vitamin B(6), and vitamin B(12) were inversely related to the ORs for cervical SILs, after adjustment for HPV DNA and other confounders. The OR among women in the highest quartile compared with women in the lowest quartile of folate intake was 0.3 (95% CI, 0.1-0.7; P for trend = 0.002). Women with the variant T allele and folate intakes below the median were at significantly elevated risk of cervical SILs (OR, 5.0; 95% CI, 2.0-12.2) compared to women with CC alleles and folate intakes above the median. HPV infection was a strong risk factor for cervical dysplasia, particularly among women with the variant T allele (OR, 46.6; 95% CI, 15.9-136.2). All associations of MTHFR genotype with the ORs for cervical SILs were independent of other risk factors under study. These findings suggest that the MTHFR T allele and reduced dietary folate may increase the risk for cervical SILs.

Case-control study of ovarian cancer and polymorphisms in genes involved in catecholestrogen formation and metabolism.

Steroid hormones, such as estrogens, appear to be associated with ovarian carcinogenesis, but the precise biological mechanisms are unclear. Polymorphisms in genes that regulate the concentration of estrogens and their metabolites may contribute directly to the individual variation in ovarian cancer risk through a mechanism involving oxidative stress or indirectly by influencing ovarian cancer susceptibility associated with ovulation and reproduction. We conducted a population-based, case-control study of primary ovarian cancer between 1993 and 1999 in Hawaii to test several genetic and related hypotheses. A personal interview and blood specimen were obtained in the subjects' homes. In a sample of 129 epithelial ovarian cancer cases and 144 controls, we compared the frequencies of several polymorphisms in genes that regulate steroid hormone metabolism and catecholestrogen formation. Multivariate unconditional logistic regression was used to model the association of each genetic polymorphism separately after adjusting for age, ethnicity, and other covariates. The high-activity Val432 allele of the CYP1B1 gene, which may be linked to oxidative stress through elevated 4-hydroxylated catecholestrogen formation, was associated with an increased risk of ovarian cancer. The Val/Leu genotype for CYP1B1 was associated with an odds ratio of 1.8 (95% confidence interval, 1.0-3.3) and the Val/Val genotype with an odds ratio of 3.8 (95% confidence interval, 1.2-11.4) compared with the Leu/Leu genotype (P = 0.005). Tobacco smokers with at least one CYP1A1 (MspI) m2 allele, one CYP1B1 Val allele, one COMT Met allele, or two CYP1A2 A alleles were at significantly increased risk of ovarian cancer compared to never-smokers with CYP1A1 (MspI) ml/ml, CYP1B1 Leu/Leu, COMT Val/Val, or CYP1A2 A/A genotypes, respectively. We found a positive statistical interaction (P = 0.03) between tobacco smoking and the CYP1A1 (MspI) polymorphism on the risk of ovarian cancer. None of the other gene-environment (pregnancy, oral contraceptive pill use) or gene-gene interactions were statistically significant. Although not significant, there was a suggestion that the effect of the CYP1B1 Val allele was reduced substantially in the presence of the high-activity COMT Met allele. These findings suggest that the CYP1B1-Val allele and perhaps other genetic polymorphisms in combination with environmental or hormonal exposures are susceptibility factors for ovarian cancer.

Correlation between growth control, neoplastic potential and endogenous connexin43 expression in HeLa cell lines: implications for tumor progression.

A HeLa cell line, obtained from the ATCC, was cloned and found to exhibit a spectrum of in vitro and in vivo growth characteristics as well as variable expression of endogenous connexin43 (Cx43), a widely expressed gap junction protein implicated in growth control. The majority of clones expressed functional Cx43, which contrasted with previous studies reporting that HeLa cells are completely negative for Cx43 mRNA/protein expression. This endogenous Cx43 expression correlated with increased growth control: Cx43-positive clones exhibited a decreased saturation density and a diminished growth capacity when in co-culture with growth-controlled normal cells in constrast to Cx43-negative clones. Endogenous Cx43 expression was negatively correlated with neoplastic potential as evidenced by attenuated anchorage-independent growth and decreased tumorigenicity in immunodeficient mice. Treatment of Cx43-negative cells with 5-aza-2'-deoxycytidine resulted in expression of Cx43, suggesting gene silencing via DNA methylation. These results support the concept of growth control via junctionally transmitted signals and suggest an epigenetic mechanism for tumor cells to circumvent this control during carcinogenesis. Moreover, the heterogeneous nature of this cell line and the ease of connexin43 gene induction suggest caution in the interpretation of results involving gene transfection using noninducible gene expression systems.

Determination of the breakpoint of the common alpha-thalassaemia deletion in Filipinos in Hawaii.

The region of the crossover causing the Filipino type of alpha thalassaemia has been determined by examining similarity between the regions which had been indicated as involved in the crossover points by restriction mapping, using the published alpha-globin region DNA sequence. The crossover point was found in 21 base pairs between two Alu sequences using PCR primers flanking these Alu sequences. A simple PCR multiplex assay has been devised to detect heterozygotes and homozygotes.

Prader-Willi syndrome is caused by disruption of the SNRPN gene.

A Prader-Willi syndrome patient is described who has a de novo balanced translocation, (4;15)(q27;q11.2)pat, with breakpoints lying between SNRPN exons 2 and 3. Parental-origin studies indicate that there is no uniparental disomy and no apparent deletion. This patient expresses ZNF127, SNRPN exons 1 and 2, IPW, and D15S227E (PAR1) but does not express either SNRPN exons 3 and 4 or D15S226E (PAR5), as assayed by reverse transcription-PCR, of peripheral blood cells. Methylation studies showed normal biparental patterns of inheritance of loci DN34/ZNF127, D15S63, and SNRPN exon 1. Results for this patient and that reported by Sun et al. support the contention that an intact genomic region and/or transcription of SNRPN exons 2 and 3 play a pivotal role in the manifestations of the major clinical phenotype in Prader-Willi syndrome.

Non-mosaic trisomy 16 in a third-trimester fetus.

BACKGROUND: Trisomy 16 in the most common trisomy first-trimester spontaneous abortions, suggesting a high rate of non-disjunction of this chromosome. Deoxyribonucleic acid studies in aborted conceptuses with trisomy 16 have demonstrated a maternal origin in all cases. There have been cases of confined placental mosaicism, fetal mosaicism, and partial trisomy involving chromosome 16 reported in term fetuses. However, to our knowledge, there have been no previous reports of a near-term fetus with full trisomy 16 since the advent of modern chromosomal banding techniques. CASE: A 25-year-old Filipino woman underwent obstetric sonographic evaluation at 32 weeks' gestation; results were remarkable for oligohydramnios, severe growth restriction, and multiple dysmorphic features. Percutaneous umbilical blood sampling was performed for rapid karyotyping, viral serology, and blood profiles. The fetal karyotype was 47, XY+16; the remainder of the laboratory analysis was unremarkable. The patient went into spontaneous labor at 35 weeks' gestation and delivered a stillborn female fetus (birth weight 783 g). Chromosomes from skin, brain, and chorionic villi were examined and all demonstrated trisomy 16 (47, XX,+16). Deoxyribonucleic acid primers for known polymorphic regions of chromosome 16 were used and determined the origin of the extra chromosome to be non-disjunction during paternal meiosis. CONCLUSION: Previously, full trisomy 16 has been thought to be incompatible with fetal survival past the early second trimester. This case also contrasts with previously reported experience with trisomy 16 in that parental origin studies determined that the extra chromosome 16 originated from the father, suggesting that paternal derivation of the additional chromosome may play a role in the ultimate phenotypic expression.

Deletion involving D15S113 in a mother and son without Angelman syndrome: refinement of the Angelman syndrome critical deletion region.

Deletions of 15q11-q13 typically result in Angelman syndrome when inherited from the mother and Prader-Willi syndrome when inherited from the father. The critical deletion region for Angelman syndrome has recently been restricted by a report of an Angelman syndrome patient with a deletion spanning less than 200 kb around the D15S113 locus. We report here on a mother and son with a deletion of chromosome 15 that includes the D15S113 locus. The son has mild to moderate mental retardation and minor anomalies, while the mother has a borderline intellectual deficit and slightly downslanting palpebral fissures. Neither patient has the seizures, excessive laughter and hand clapping, ataxia or the facial anomalies which are characteristic of Angelman syndrome. The proximal boundary of the deletion in our patients lies between the D15S10 and the D15S113 loci. Our patients do not have Angelman syndrome, despite the deletion of the D15S113 marker. This suggests that the Angelman syndrome critical deletion region is now defined as the overlap between the deletion found in the previously reported Angelman syndrome patient and the region that is intact in our patients.

Characterization of spontaneous factor-independent cell lines derived from the human leukemic cell line TF-1: a dominant event.

Uncontrolled proliferation of acute myeloid leukemia (AML) cells is an important step during leukemogenesis. However, little is known about the mechanisms leading to growth autonomy. Studies using immortalized murine hematopoietic cell lines have suggested that autocrine production of growth factors, or the constitutive activation of molecules in growth factor signalling pathways, are involved. We have established six spontaneous factor-independent cell lines from the human growth factor-dependent TF-1 cell line. The factor-independent cells showed no detectable growth factor activity. Immunoblotting analyses of tyrosine phosphorylation, Raf-1 and extracellular signal-regulated kinase 2 (ERK-2) showed a similar pattern in all the cell lines including TF-1 cells. Furthermore, somatic-cell hybrids between TF-1 and the factor-independent cells grew in absence of growth factor. Taken together this data demonstrates that the factor independence in this system is dominant and suggests that the molecular event is located either downstream of the Raf-1 and MAP kinases pathway or on an alternative pathway. Finally, the karyotype analysis of one factor-independent cell line TF-1i1 and TF-1H- (G418 resistant, HAT sensitive TF-1 cells) and their hybrids demonstrated an unstable derivative chromosome [der(19) t(19;?) (q13.1;?)] which seemed to correlate with the factor-independence capacity. This model may help in our understanding of autonomous proliferation by human myeloid leukemias.

Enhanced detection of the t(14;18) translocation in malignant lymphoma using pulsed-field gel electrophoresis.

The t(14;18) chromosomal translocation that results in the juxtaposition of the bcl-2 proto-oncogene with the heavy chain JH locus is a common cytogenetic abnormality in human lymphoma. In particular, it is seen in about 85% of follicular lymphoma (FL) and up to one-third of diffuse lymphomas (DL). The chromosome 18 breakpoints have been shown to cluster into two regions. The major breakpoint region (mbr) within the 3' untranslated region of the bcl-2 proto-oncogene accounts for approximately 60% of the cases and the minor cluster region (mcr) 30 kb 3' of bcl-2 accounts for approximately 25% of the breakpoints. Because of variability in the position of the breakpoint, detection of the t(14;18) by Southern blot analysis provides an important clonal marker for the tumor. However, conventional electrophoresis (CE) fails to detect the translocation in 15% to 25% of cases. We have applied pulsed-field gel electrophoresis (PFGE) to the detection of the t(14;18) in a series of lymphoma prospectively analyzed by CE, polymerase chain reaction (PCR), and cytogenetic analysis. PFGE readily detected t(14;18) rearrangements as indicated by comigration of bands detected with probes for the mbr region (chromosome 18) and the JH locus (chromosome 14). In a series of 40 patients with FL, this method proved to be the most comprehensive for detection of the translocation compared with standard methods; in fact, in one case only PFGE was able to detect the chromosomal rearrangement. Ten percent of the FL cases were negative by all methods tested. In a separate analysis of matched tissue specimens from cases of tumor progression of FL to diffuse lymphoma, PFGE detected a common t(14;18) rearrangement confirming a clonal origin in seven of seven cases, whereas CE detected a rearrangement in only three of seven cases. Overall, PFGE was able to detect a translocation in 8 of 12 cases that were negative by CE and four of eight negative by cytogenetic analysis. In conclusion, PFGE analysis is more comprehensive than CE, PCR, and cytogenetic analysis for the detection of the t(14;18) breakpoint in tissue biopsies of malignant lymphoma.

Importance of bone marrow cytogenetic evaluation before autologous bone marrow transplantation for Hodgkin's disease.

Alkylating agents used either with or without radiation therapy have been associated with the development of myelodysplastic syndrome (MDS) and acute nonlymphoblastic leukemia (ANLL) after treatment of both malignant and nonmalignant disorders. This report describes seven patients with recurrent Hodgkin's disease (HD) evaluated for bone marrow transplantation (BMT) who developed chromosomal abnormalities, and emphasizes the importance of bone marrow cytogenetic studies before bone marrow harvest. Three patients with histologically normal bone marrow underwent autologous BMT and subsequently developed an MDS or ANLL. Four patients had the clonal abnormality detected before bone marrow harvest and did not proceed to BMT.

Deletion mapping of DNA segments from the Y chromosome long arm and their analysis in an XX male.

Twelve DNA segments have been localized to the long arm of the Y chromosome and were assigned to three intervals by deletion mapping. Of these segments, six were from distal Yq11.23, which is supposed to contain a spermatogenesis locus. The physical mapping information was used to analyze an XX male who is positive for DNA sequences both from distal Yp and from Yq. Two of the twelve sequences from Yq (Y-198 and Y-253) were detected in this patient along with two of six short-arm segments tested. Long-range physical mapping placed Y-198 and Y-253 on a common 1100-kb BssHII fragment. In this patient, the long-arm sequences were assigned to distal Xp by in situ hybridization. The data suggest that this XX male derived from an unequal interchange between an X and an inverted Y chromosome presumed to have been present in the patient's father.

Detection of bcr-abl fusion in chronic myelogeneous leukemia by in situ hybridization.

Chronic myelogeneous leukemia (CML) is genetically characterized by fusion of the bcr and abl genes on chromosomes 22 and 9, respectively. In most cases, the fusion involves a reciprocal translocation t(9;22)(q34;q11), which produces the cytogenetically distinctive Philadelphia chromosome (Ph1). Fusion can be detected by Southern (DNA) analysis or by in vitro amplification of the messenger RNA from the fusion gene with polymerase chain reaction (PCR). These techniques are sensitive but cannot be applied to single cells. Two-color fluorescence in situ hybridization (FISH) was used with probes from portions of the bcr and abl genes to detect the bcr-abl fusion in individual blood and bone marrow cells from six patients. The fusion event was detected in all samples analyzed, of which three were cytogenetically Ph1-negative. One of the Ph1-negative samples was also PCR-negative. This approach is fast and sensitive, and provides potential for determining the frequency of the abnormality in different cell lineages.

Expression of bcr-abl fusion transcripts following bone marrow transplantation for Philadelphia chromosome-positive leukemia.

A modified polymerase chain reaction (PCR) procedure was used to study the expression of bcr-abl fusion transcripts following allogeneic bone marrow transplantation (BMT) for Philadelphia chromosome (Ph1) positive acute and chronic leukemias. The technique was applied to RNA preparations of peripheral blood and bone marrow cells from 10 patients with chronic myelogenous leukemia (CML) and one patient with acute lymphoblastic leukemia (ALL), all of whom had undergone allogenic BMT and were in clinical and cytogenetic remission. Pre-BMT samples available for eight of 11 patients contained detectable bcr-abl fusion products serving as a baseline for comparison to post-BMT studies. Six patients showed no PCR-detectable bcr-abl transcripts in each of several serial analyses post-BMT (1-36 months post-BMT). The remaining five patients demonstrated various patterns of bcr-abl transcript expression after transplantation. In three patients, bcr-abl transcripts persisted for up to 3 months post-BMT but subsequently were undetectable. Molecular relapse was observed 3 and 6 months post-BMT in the remaining two patients whose earlier post-BMT samples showed no bcr-abl fusion transcripts. No bcr-abl transcripts were detected in subsequent samples from both of these patients 6 months and 1 year post-BMT, respectively. These data confirm that Ph1 carrying cells expressing the bcr-abl fusion mRNA may persist or recur for several months following BMT in the absence of clinical and cytogenetic relapse. The significance of these observations is discussed with respect to results reported recently by others using similar techniques.

Induction of proliferation of human follicular (B type) lymphoma cells by cognate interaction with CD4+ T cell clones.

We examined stimuli which are required for the induction of in vitro proliferation of follicular lymphoma cells, a low grade non-Hodgkin's B cell lymphoma characterized by a specific chromosomal translocation, t(14;18)(q32;q21), and by in vivo growth of the lymphoma cells in germinal center-like follicles infiltrated with CD4+ T cells. The purified follicular lymphoma cells, which are morphologically uniform, small, and dense, did not respond to stimulation with soluble lymphokines in the absence of T cells. Vigorous in vitro proliferation of follicular lymphoma cells was induced, however, when the follicular lymphoma cells were cultured with a CD4+ T cell clone which recognized alloantigens expressed by the lymphoma cells. This response required B-T cell contact, and was inhibited by anti-class II but not by anti-class I MHC mAb, indicating that these neoplastic B cells behaved as normal B cells and responded to normal activation and differentiation signals from T cells. After the cognate B lymphoma-T cell interaction occurred in culture, addition of IL-2 or IL-4 enhanced the proliferation of the tumor cells. These results, with a monoclonal and homogeneous population of B cells, affirm the idea that cognate interaction between B cells and Th cells is required for the effective activation of resting B cells. Moreover, these results suggest that a critical host-tumor interaction occurs in vivo, and that the polyclonal CD4+ T cells that infiltrate follicular lymphomas play a role in sustaining rather than inhibiting tumor growth in vivo. If so, therapies directed not only against the neoplastic cell but also against specific T cells and their cognate interactions with tumor cells may have a rationale.

The gene for the RNA component of the mitochondrial RNA-processing endoribonuclease is located on human chromosome 9p and on mouse chromosome 4.

Mitochondrial RNA-processing endoribonuclease (RNAase MRP) has the capacity to cleave mitochondrial RNA complementary to the light strand of the displacement loop at a unique site. The enzyme is a ribonucleoprotein whose RNA component is a nuclear gene product. The 5' flanking region of the primary transcript has control elements characteristic of RNA polymerase II transcription, and the coding region has features of RNA polymerase III transcription signals. The RNA associated with RNAase MRP is the first known RNA encoded by a single-copy gene in the nucleus and believed to be imported into mitochondria. The gene (RMRP) for this RNA component of RNAase MRP was assigned to human chromosome 9 and mouse chromosome 4 by Southern blot analyses of 11 human X rodent hybrids and 11 mouse X rodent hybrids with probe pHM1.0 and probe pSP270, respectively. In situ hybridization of probe pHSTU300 to normal human chromosomes revealed 29 of 100 cells with label on 9p and 9.6% of 302 silver grains located at 9p21--p12.

Localization of a human T-cell-specific gene, RANTES (D17S136E), to chromosome 17q11.2-q12.

We report here the localization of the gene for a human T-cell-specific molecule, designated RANTES, to human chromosome region 17q11.2-q12 by in situ hybridization and analysis of somatic cell hybrids using a cDNA probe to the gene. We have recently shown that this gene, which encodes a small, secreted, putative lymphokine, is a member of a larger gene family some of whose members reside on chromosome 4 but most of whose members have not to date been mapped. A secondary hybridization peak was noted on the region of human chromosome 5q31-q34, which may represent the location of other members of the gene family. Interestingly, this latter region overlaps with the location of an extended linked cluster of growth factor and receptor genes, some of which may be coregulated with members of the RANTES gene family.