ABSTRACT
Glioblastoma (GBM) is an aggressive and incurable tumor of the brain with limited treatment options. Current first-line standard of care is the DNA alkylating agent temozolomide (TMZ), but this treatment strategy adds only ~4 months to median survival due to the rapid development of resistance. While some mechanisms of TMZ resistance have been identified, they are not fully understood. There are few effective strategies to manage therapy resistant GBM, and we lack diverse preclinical models of acquired TMZ resistance in which to test therapeutic strategies on TMZ resistant GBM. In this study, we create and characterize two new GBM cell lines resistant to TMZ in vitro, based on the 8MGBA and 42MGBA cell lines. Analysis of the TMZ resistant (TMZres) variants in conjunction with their parental, sensitive cell lines shows that acquisition of TMZ resistance is accompanied by broad phenotypic changes, including increased proliferation, migration, chromosomal aberrations, and secretion of cytosolic lipids. Importantly, each TMZ resistant model captures a different facet of the "go" (8MGBA-TMZres) or "grow" (42MGBA-TMZres) hypothesis of GBM behavior. These in vitro model systems will be important additions to the available tools for investigators seeking to define molecular mechanisms of acquired TMZ resistance.
Subject(s)
Actin Cytoskeleton/drug effects , Antineoplastic Agents, Alkylating/pharmacology , Cell Proliferation/drug effects , Drug Resistance, Neoplasm/drug effects , Gene Expression Regulation, Neoplastic , Temozolomide/pharmacology , Actin Cytoskeleton/metabolism , Actin Cytoskeleton/ultrastructure , Apoptosis/drug effects , Brain Neoplasms/drug therapy , Brain Neoplasms/genetics , Brain Neoplasms/metabolism , Brain Neoplasms/pathology , Carmustine/pharmacology , Cell Cycle/drug effects , Cell Cycle/genetics , Cell Line, Tumor , Cell Movement/drug effects , Cell Size , Chromosome Duplication , DNA Modification Methylases/genetics , DNA Modification Methylases/metabolism , DNA Repair Enzymes/genetics , DNA Repair Enzymes/metabolism , Drug Resistance, Neoplasm/genetics , Glioblastoma/drug therapy , Glioblastoma/genetics , Glioblastoma/metabolism , Glioblastoma/pathology , Humans , Metabolic Networks and Pathways/drug effects , Metabolic Networks and Pathways/genetics , Metabolome/drug effects , Models, Biological , Neoplasm Proteins/genetics , Neoplasm Proteins/metabolism , Tumor Suppressor Proteins/genetics , Tumor Suppressor Proteins/metabolismABSTRACT
Metastasis of cancer cells involves multiple steps, including their dissociation from the primary tumor and invasion through the endothelial cell barrier to enter the circulation and finding their way to distant organ sites where they extravasate and establish metastatic lesions. Deficient contact inhibition is a hallmark of invasive cancer cells, yet surprisingly the vascular invasiveness of commonly studied cancer cell lines is regulated by the density at which cells are propagated in culture. Cells grown at high density were less effective at invading an endothelial monolayer than cells grown at low density. This phenotypic difference was also observed in a zebrafish model of vascular invasion of cancer cells after injection into the yolk sac and extravasation of cancer cells into tissues from the vasculature. The vascular invasive phenotypes were reversible. A kinome-wide RNA interference screen was used to identify drivers of vascular invasion by panning small hairpin RNA (shRNA) library-transduced noninvasive cancer cell populations on endothelial monolayers. The selection of invasive subpopulations showed enrichment of shRNAs targeting the large tumor suppressor 1 (LATS1) kinase that inhibits the activity of the transcriptional coactivator yes-associated protein (YAP) in the Hippo pathway. Depletion of LATS1 from noninvasive cancer cells restored the invasive phenotype. Complementary to this, inhibition or depletion of YAP inhibited invasion in vitro and in vivo. The vascular invasive phenotype was associated with a YAP-dependent upregulation of the cytokines IL6, IL8 and C-X-C motif ligand 1, 2 and 3. Antibody blockade of cytokine receptors inhibited invasion and confirmed that they are rate-limiting drivers that promote cancer cell vascular invasiveness and could provide therapeutic targets.
Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Cell Movement , Endothelium, Vascular/pathology , Gene Expression Regulation, Neoplastic , Phosphoproteins/metabolism , Protein Serine-Threonine Kinases/metabolism , Receptors, Interleukin-8B/metabolism , Adaptor Proteins, Signal Transducing/genetics , Animals , Apoptosis , Blotting, Western , Breast Neoplasms/genetics , Cell Cycle , Cell Proliferation , Cytokines/genetics , Cytokines/metabolism , Endothelium, Vascular/metabolism , Female , Human Umbilical Vein Endothelial Cells , Humans , Mice , Mice, Nude , Neoplasm Invasiveness , Phosphoproteins/genetics , Phosphorylation , Protein Serine-Threonine Kinases/antagonists & inhibitors , Protein Serine-Threonine Kinases/genetics , RNA Interference , RNA, Messenger/genetics , Real-Time Polymerase Chain Reaction , Receptors, Interleukin-8B/genetics , Reverse Transcriptase Polymerase Chain Reaction , Transcription Factors , Tumor Cells, Cultured , Xenograft Model Antitumor Assays , YAP-Signaling Proteins , ZebrafishABSTRACT
The BH3-like motif-containing inducer of cell death (BLID) is an intronless gene localized on 11q24.1. Loss of that region has frequently been reported in early-onset breast cancer and is significantly associated with poor prognosis and reduced survival. Downregulation of BLID is associated with younger age, triple-negative phenotype, and reduced disease-free and overall survival of breast cancer patients. In this study, we investigated allelic loss of BLID in breast tumor specimens from 78 women with invasive breast cancer using 2 dinucleotide polymorphic markers closely linked to the BLID gene (no intragenic marker for BLID is available). Seventy-three cases were informative. Overall, loss of heterozygosity (LOH) at the BLID locus was detected in 32% of the informative cases (23/73). However, in patients 40 years old and younger, LOH was detected in 50% of the cases (9/18). Patients aged 40 years and younger were significantly more likely to experience LOH than those aged 41-55 years (p = 0.04). Specifically, the odds of BLID loss for patients aged 40 years and younger were 3.7 times the odds of loss for patients aged 41-55 years (95% CI, 1.1-13). Our findings suggest a tumor suppressor role of the BLID gene in early-onset breast cancer.
Subject(s)
Apoptosis Regulatory Proteins , Biomarkers, Tumor/analysis , Breast Neoplasms , Chromosome Mapping/methods , Cytogenetics/methods , DNA, Neoplasm/analysis , Adult , Age Factors , Age of Onset , Aged , Alleles , Apoptosis Regulatory Proteins/deficiency , Apoptosis Regulatory Proteins/genetics , Biomarkers, Tumor/genetics , Breast Neoplasms/diagnosis , Breast Neoplasms/genetics , Breast Neoplasms/metabolism , Breast Neoplasms/mortality , Breast Neoplasms/pathology , DNA Primers/chemistry , DNA Primers/genetics , DNA Probes/chemistry , DNA Probes/genetics , DNA, Neoplasm/genetics , Female , Humans , Loss of Heterozygosity , Middle Aged , Neoplasm Invasiveness , Odds Ratio , Prognosis , Software , Survival RateABSTRACT
Holoprosencephaly (HPE), which results from failed or incomplete midline forebrain division early in gestation, is the most common forebrain malformation. The etiology of HPE is complex and multifactorial. To date, at least 12 HPE-associated genes have been identified, including TGIF (transforming growth factor beta-induced factor), located on chromosome 18p11.3. TGIF encodes a transcriptional repressor of retinoid responses involved in TGF-ß signaling regulation, including Nodal signaling. TGIF mutations are reported in approximately 1-2% of patients with non-syndromic, non-chromosomal HPE. We combined data from our comprehensive studies of HPE with a literature search for all individuals with HPE and evidence of mutations affecting TGIF in order to establish the genotypic and phenotypic range. We describe 2 groups of patients: 34 with intragenic mutations and 21 with deletions of TGIF. These individuals, which were ascertained from our research group, in collaboration with other centers, and through a literature search, include 38 probands and 17 mutation-positive relatives. The majority of intragenic mutations occur in the TGIF homeodomain. Patients with mutations affecting TGIFrecapitulate the entire phenotypic spectrum observed in non-chromosomal, non-syndromic HPE. We identified a statistically significant difference between the 2 groups with respect to inheritance, as TGIF deletions were more likely to be de novo in comparison to TGIF mutations (χ(2) ((2)) = 6.97, p(permutated) = 0.0356). In addition, patients with TGIF deletions were also found to more commonly present with manifestations beyond the craniofacial and neuroanatomical features associated with HPE (p = 0.0030). These findings highlight differences in patients with intragenic mutations versus deletions affecting TGIF, and draw attention to the homeodomain region, which appears to be particularly relevant to HPE. These results may be useful for genetic counseling of affected patients.
ABSTRACT
The sentinel lymph node (SLN) is considered to be the first axillary node that contains malignant cells in metastatic breast tumors, and its positivity is currently used in clinical practice as an indication for axillary lymph node dissection. Therefore, accurate evaluation of the SLN for the presence of breast metastatic cells is essential. The main aim of our study is to characterize the genomic changes present in the SLN metastatic samples with the ultimate goal of improving the predictive value of SLN evaluation. Twenty paired samples of SLN metastases and their corresponding primary breast tumors (PBT) were investigated for DNA copy number changes using comparative genomic hybridization (CGH). Non-random DNA copy number changes were observed in all the lesions analyzed, with gains being more common than losses. In 75% of the cases there was at least one change common to both PBT and SLN. The most frequent changes detected in both lesions were gains of 1pter-->p32, 16, 17, 19, and 20 and losses of 6q13-->q23 and 13q13-->q32. In the PBT group, alterations on chromosomes 1, 16, and 20 were the most frequent, whereas chromosomes 1, 6, and 19 were the ones with the highest number of changes in the SLN metastatic group. A positive correlation was found between the DNA copy number changes per chromosome in each of the groups. Our findings indicate the presence of significant DNA copy number changes in the SLN metastatic lesions that could be used in the future as additional markers to improve the predictive value of SLN biopsy procedure.
Subject(s)
Breast Neoplasms/genetics , DNA, Neoplasm/genetics , Lymphatic Metastasis/genetics , Adult , Aged , Breast Neoplasms/secondary , Chromosome Mapping , DNA, Neoplasm/analysis , Female , Gene Dosage , Humans , Karyotyping , Middle Aged , Oligonucleotide Array Sequence Analysis , Sentinel Lymph Node BiopsyABSTRACT
A supernumerary ring chromosome was found on amniocentesis performed for advanced maternal age. A review of the literature found 34 reports of supernumerary ring chromosome I which are compared to our case.
Subject(s)
Amniocentesis , Chromosome Aberrations , Chromosomes, Human, Pair 1/genetics , Genetic Markers/genetics , Ring Chromosomes , Abortion, Eugenic , Adult , Female , Genetic Counseling , Genetic Testing , Humans , In Situ Hybridization, Fluorescence , Karyotyping , Male , Maternal Age , PregnancyABSTRACT
Holoprosencephaly (HPE) is the most common structural malformation of the developing forebrain. At birth, nearly 50% of children with HPE have cytogenetic anomalies. Approximately 20% of infants with normal chromosomes have sequence mutations in one of the four main HPE genes (SHH, ZIC2, SIX3, and TGIF). The other non-syndromic forms of HPE may be due to environmental factors or mutations in other genes, or potentially due to submicroscopic deletions of HPE genes. We used two complementary assays to test for HPE associated submicroscopic deletions. Firstly, we developed a multicolour fluorescent in situ hybridisation (FISH) assay using probes for the four major HPE genes and for two candidate genes (DISP1 and FOXA2). We analysed lymphoblastoid cell lines (LCL) from 103 patients who had CNS findings of HPE, normal karyotypes, and no point mutations, and found seven microdeletions. We subsequently applied quantitative PCR to 424 HPE DNA samples, including the 103 samples studied by FISH: 339 with CNS findings of HPE, and 85 with normal CNS and characteristic HPE facial findings. Microdeletions for either SHH, ZIC2, SIX3, or TGIF were found in 16 of the 339 severe HPE cases (that is, with CNS findings; 4.7%). In contrast, no microdeletion was found in the 85 patients at the mildest end of the HPE spectrum. Based on our data, microdeletion testing should be considered as part of an evaluation of holoprosencephaly, especially in severe HPE cases.
Subject(s)
Holoprosencephaly/genetics , In Situ Hybridization, Fluorescence/methods , Sequence Deletion , Base Sequence , Cell Line , Eye Proteins/genetics , Genetic Testing , Hedgehog Proteins , Hepatocyte Nuclear Factor 3-beta/genetics , Holoprosencephaly/diagnosis , Homeodomain Proteins/genetics , Humans , Karyotyping , Nerve Tissue Proteins/genetics , Nuclear Proteins , Polymerase Chain Reaction , Repressor Proteins/genetics , Trans-Activators/genetics , Transcription Factors/genetics , Homeobox Protein SIX3ABSTRACT
AIM: The evaluation of allelic losses at the FHIT and the BRCA1 genes and at three other loci at the 17q region in a series of 34 sporadic breast cancer cases from Southern Brazil. METHODS: The samples were evaluated for loss of heterozygosity (LOH) at the FHIT and the BRCA1 genes and at three other microsatellite markers at 17q, and the findings were correlated with clinicopathological parameters. RESULTS: The BRCA1 intragenic marker, D17S855, had the highest frequency of LOH, detected in 10 of 24 informative cases, followed by the D17S579 (six of 23 informative cases), D17S806 (five of 21 informative cases), and D17S785 markers (five of 21 informative cases). LOH at the FHIT intragenic marker, D3S1300, was found in six of 25 informative cases. In four of the six cases with LOH of the FHIT gene, there was concomitant loss of the BRCA1 intragenic marker. CONCLUSIONS: The frequency of allelic losses in the FHIT and BRCA1 loci in the Southern Brazilian population is similar to that described in the general population. No correlations were found when the total LOH frequency was compared with tumour size, grade, or presence of axillary lymph node metastasis. Further studies using larger sporadic breast cancer samples and additional markers would be useful to confirm these findings, in addition to establishing more specific associations with clinicopathological parameters in this specific population.
Subject(s)
Acid Anhydride Hydrolases , Breast Neoplasms/genetics , Carcinoma, Ductal, Breast/genetics , Genes, BRCA1 , Loss of Heterozygosity , Neoplasm Proteins/genetics , Adult , Aged , Brazil , Breast Neoplasms/pathology , Carcinoma, Ductal, Breast/pathology , Chi-Square Distribution , Female , Gene Frequency , Genetic Markers , Humans , Lymphatic Metastasis , Middle Aged , Polymerase Chain Reaction/methodsABSTRACT
Classic cytogenetic and comparative genomic hybridisation (CGH) data on osteosarcomas have been reported extensively in the literature. However, the number of paediatric osteosarcoma cases studied below the age of 14 years remains relatively small. This study reports four new cases of paediatric osteosarcoma in patients aged 3 to 13 years, evaluated by classic cytogenetics and CGH analyses. Clonal chromosomal alterations were detected in all the cases and included structural rearrangements at 1p11-13, 1q11, 4q27-33, 6p23-25, 6q16-25, 7p13-22, 7q11-36, 11p10-15, 11q23, 17p11.2-13, 21p11, and 21q11-22. The CGH analysis revealed recurrent gains at 1p, 4q, 17p, and 21q and losses at 3q and 16p. Five amplification sites were observed at 1q11-23, 6p21, 8q13, 8q21.3-24.2, and 17p. The data are discussed and compared with other cytogenetic reports in the literature.
Subject(s)
Bone Neoplasms/genetics , Chromosome Aberrations , Osteosarcoma/genetics , Adolescent , Child , Child, Preschool , Cytogenetic Analysis/methods , Female , Humans , Karyotyping , Male , Nucleic Acid HybridizationABSTRACT
We report a case of a lipoblastoma in a 10-month-old girl in which the cytogenetic aberration showed a homogeneously staining-like region (hsr) within two derivative chromosomes 8. There was a loss of one normal copy of chromosome 8 and gain of two identical derivative chromosomes 8 with the karyotype designation 47,XX,psu idic(8)(pter-->q12 approximately 13::hsr::q12 approximately 13-->pter),+psu idic (8)(pter-->q12 approximately 13::hsr::q12 approximately 13-->pter). This is the first report of a chromosomal aberration of this type seen in lipoblastoma.
Subject(s)
Chromosomes, Human, Pair 8 , Lipoma/genetics , Chromosome Banding , Female , Humans , In Situ Hybridization, Fluorescence , Infant , KaryotypingABSTRACT
OBJECTIVE: To evaluate the detection of aneuploidy in archival tissues from miscarriages by a method that uses microdissection and DNA extraction of villus cells from paraffin blocks, followed by universal DNA amplification and comparative genomic hybridization (CGH). DESIGN: Retrospective analysis. SETTING: Academic medical center. PATIENT(S): Nine archival tissues from cases of spontaneous abortion with trisomy 16 (two cases), trisomy 21 (three cases), trisomy 22 (two cases), triploidy (one case), and monosomy X (one case). INTERVENTION(S): Villus DNA was extracted from microdissected, formalin-fixed, paraffin-embedded tissues. Aneuploidy was detected by CGH after universal amplification of the DNA with the use of degenerate oligonucleotide-primed polymerase chain reaction. MAIN OUTCOME MEASURE(S): Detection of aneuploidy in archival pregnancy-loss tissues using CGH. RESULT(S): In all nine cases, DNA was successfully extracted from the microdissected tissues and was of sufficient quantity and quality for evaluation by CGH. In six of nine cases, the chromosomal abnormality detected by conventional cytogenetic analysis was identified by CGH: trisomy 16 (2/2), trisomy 21 (3/3), and trisomy 22 (1/2). One case of each of the following was not detectable: triploidy (1/1), monosomy X (1/1), and trisomy 22 (1/2). CONCLUSION(S): We propose CGH as a method for determination of aneuploidy in pregnancy-loss archival tissues when conventional cytogenetic analysis is unsuccessful or when it was not performed when fresh tissue was available.
Subject(s)
Abortion, Spontaneous/genetics , Aneuploidy , Chromosome Aberrations , Archives , Chromosomes, Human, Pair 16 , Chromosomes, Human, Pair 22 , Cytogenetic Analysis , DNA/analysis , DNA/isolation & purification , Down Syndrome/genetics , Female , Fluorescent Dyes , Humans , Nucleic Acid Hybridization , Paraffin , Polymerase Chain Reaction , Pregnancy , Retrospective Studies , Tissue Embedding , Trisomy , X ChromosomeABSTRACT
The authors report on a young girl with generalized developmental deficits originally thought to be caused by an unusual reaction to DPT vaccination. At the age of 4(1/2) years, chromosome analysis showed that the terminus of the short arm of chromosome 9 had extra material believed to originate from 7p terminus, thus she was considered to be trisomic for a segment of 7p and monosomic for a small portion of 9p [46,XX,der (9), t(7;9)(p15;p24)]. Ten years later, molecular cytogenetic testing using fluorescence in situ hybridization (FISH) confirmed that the extra chromosomal material represented partial trisomy 7p. The proposita had a high and large forehead, hypertelorism, and broad nasal bridge, findings seen in most individuals with trisomy 7p. Long-term follow-up showed the presence of hypothyroidism, obesity, and cerebral palsy. A review of all published cases of trisomy 7p with focus on associated complications suggests a well-defined pattern of abnormalities characterized by musculoskeletal, cardiovascular, neurological, genital, and ocular abnormalities in decreasing frequency. At least one-third of affected individuals died in infancy and close to half had severe mental retardation. FISH was essential in the confirmation of the cytogenetic abnormality and further delineation of the chromosomal disorder.
Subject(s)
Chromosomes, Human, Pair 7/genetics , Chromosomes, Human, Pair 9/genetics , Translocation, Genetic/genetics , Trisomy/genetics , Adolescent , Cerebral Palsy/genetics , Child, Preschool , Female , Growth Disorders/diagnostic imaging , Growth Disorders/genetics , Humans , Hypothyroidism/genetics , In Situ Hybridization, Fluorescence , Karyotyping , Male , Obesity/genetics , Tomography, X-Ray Computed , Trisomy/pathologyABSTRACT
Depletion of poly(ADP-ribose) polymerase (PARP) increases the frequency of recombination, gene amplification, sister chromatid exchanges, and micronuclei formation in cells exposed to genotoxic agents, implicating PARP in the maintenance of genomic stability. Flow cytometric analysis now has revealed an unstable tetraploid population in immortalized fibroblasts derived from PARP(-/-) mice. Comparative genomic hybridization detected partial chromosomal gains in 4C5-ter, 5F-ter, and 14A1-C1 in PARP(-/-)mice and immortalized PARP(-/-)fibroblasts. Neither the chromosomal gains nor the tetraploid population were apparent in PARP(-/-) cells stably transfected with PARP cDNA [PARP(-/-)(+PARP)], indicating negative selection of cells with these genetic aberrations after reintroduction of PARP cDNA. Although the tumor suppressor p53 was not detectable in PARP(-/-) cells, p53 expression was partially restored in PARP(-/-) (+PARP) cells. Loss of 14D3-ter that encompasses the tumor suppressor gene Rb-1 in PARP(-/-) mice was associated with a reduction in retinoblastoma(Rb) expression; increased expression of the oncogene Jun was correlated with a gain in 4C5-ter that harbors this oncogene. These results further implicate PARP in the maintenance of genomic stability and suggest that altered expression of p53, Rb, and Jun, as well as undoubtedly many other proteins may be a result of genomic instability associated with PARP deficiency.
Subject(s)
Chromosome Aberrations , Poly(ADP-ribose) Polymerases/genetics , Transfection , Animals , Cell Line, Transformed , DNA, Complementary , Female , Gene Expression Regulation, Enzymologic/genetics , Genes, Retinoblastoma , Genes, jun , Genes, p53 , Genome , Mice , Mice, Inbred C57BL , Mice, Knockout , Nucleic Acid HybridizationABSTRACT
We describe a 19-year-old patient with a de novo mosaic add(3) chromosome (extra material of unknown origin on the 3q). The use of spectral karyotyping and fluorescence in situ hybridization using subtelomeric probes permitted the full characterization of the cytogenetic abnormality. The additional material on 3q was found to originate from 14q31-qter. This is one of the few reported cases with trisomy 14q31-qter and is the first mosaic case.
Subject(s)
Chromosomes, Human, Pair 13/genetics , Karyotyping , Mosaicism , Trisomy , Adult , Chromosome Banding , Developmental Disabilities/genetics , Female , Genotype , Humans , In Situ Hybridization, Fluorescence , Intellectual Disability/genetics , PhenotypeABSTRACT
Although several genes have been investigated in adrenal tumorigenesis, the genetic background of adrenocortical tumors (ACT) remains poorly characterized. In southern Brazil, the annual incidence of ACT is unusually high, ranging from 3.4-4.2/million children, compared with a worldwide incidence of 0.3/million children younger than 15 yr. Environmental factors have been implicated because the distribution of these tumors follows a regional, rather than a familial, pattern. However, decreased penetrance of a particular gene defect cannot be excluded. Because linkage or other traditional genetic analyses would not be appropriate to investigate the defect(s) associated with ACT in this population, we used comparative genomic hybridization (CGH) to screen for DNA sequence copy number changes in 9 nonfamilial ACT (6 carcinomas and 3 adenomas) from unrelated patients from this region. Six female (aged 10 months to 6 3/4 yr) and 3 male (1 1/12 to 3 1/4 yr) patients were studied. Three carcinomas were at stage I, 1 was at stage II, and another was at stage III. Two carcinomas had evidence of invasion of the vena cava, and 3 were more than 3 cm in size. All patients underwent surgical excision of their tumors; chemotherapy was administered to cancer patients. Currently, all patients are alive and in remission, with the exception of 1 patient with stage III cancer. High mol wt DNA was extracted from tumor tissue obtained at surgery and frozen at -70 C. This DNA was labeled and used for CGH according to standard procedures. Digital image analysis was performed to detect chromosomal gains or losses. CGH evaluation revealed extensive genetic aberrations in both adenomas and carcinomas; there were no significant differences relative to age, gender, size, or stage of the tumor (P > 0.1). Chromosomes and chromosomal regions 1q, 5p, 5q, 6p, 6q, 8p, 8q, 9q, 10p, 11q, 12q, 13q, 14q, 15q, 16, 18q, 19, and 20q demonstrated gains, whereas 2q, 3, 4, 9p, 11, 13q, 18, 20p, and Xq showed losses. The most striking finding was consistent copy number gain of chromosomal region 9q34 in 8 of the 9 tumors. We conclude that both benign and malignant ACT from southern Brazil show multiple genetic aberrations, including a consistent gain of chromosomal region 9q34. This genomic area may harbor genetic defects that predispose to ACT formation and are shared by the patients who were investigated in this study or are accumulated epigenetically under the influence of a common factor, such as an environmental mutagen.
Subject(s)
Adrenal Cortex Neoplasms/genetics , Brazil , Child, Preschool , Chromosome Aberrations/genetics , Chromosomes, Human, Pair 12/genetics , Chromosomes, Human, Pair 4/genetics , Chromosomes, Human, Pair 9/genetics , Female , Gene Dosage , Humans , Infant , Male , Nucleic Acid HybridizationABSTRACT
OBJECTIVE: To use the molecular identification of Y chromosome material in products of conception cytogenetically diagnosed as "46,XX" to confirm the occurrence of inaccurate cytogenetic test results most likely attributable to maternal cell contamination. DESIGN: Retrospective analysis. SETTING: Academic medical center. PATIENT(S): Thirty-four archival tissues from cases of spontaneous abortion with a "46,XX" karyotype based on cytogenetic analysis. INTERVENTION(S): Maternal and villus DNA were extracted from microdissected, formalin-fixed, paraffin-embedded archival tissues. The presence of the X and Y chromosomes was detected with the use of polymerase chain reaction assays and confirmed with fluorescence in situ hybridization. MAIN OUTCOME MEASURE(S): Accuracy of cytogenetic evaluation of products of conception. RESULT(S): Four (29%) of 14 first trimester and 1 (5%) of 20 second trimester "46,XX" pregnancy losses contained Y chromosome-specific DNA and demonstrated a single X chromosome-specific allele by polymerase chain reaction analysis consistent with an "XY" karyotype. Fluorescence in situ hybridization was confirmatory in 4 of 5 samples that demonstrated single X and Y signals in villus cells. CONCLUSION(S): Inaccuracy exists in the cytogenetic analysis of early products of conception that most likely is due to maternal cell contamination. In the absence of confirmatory testing, such as with a "DNA fingerprinting" assay, reports of a "46,XX" karyotype should be used cautiously in patient counseling and management.
Subject(s)
Abortion, Spontaneous/genetics , Genetic Testing/methods , Abortion, Spontaneous/diagnosis , Female , Humans , In Situ Hybridization, Fluorescence , Karyotyping , Polymerase Chain Reaction , Predictive Value of Tests , Pregnancy , Pregnancy Trimester, First , Retrospective StudiesABSTRACT
Despite major advances in molecular cytogenetics during the past decade and the important diagnostic role that fluorescence in situ hybridization (FISH) plays in the characterization of chromosomal abnormalities, the usefulness of this technique remains limited by the number of spectrally distinguishable fluorochromes or fluorochrome combinations. Overcoming this major limitation would allow one to use FISH to screen the whole human genome for chromosomal aberrations which, until recently, was possible only through conventional karyotyping. A recently described molecular cytogenetics technology, 24-color FISH using spectral karyotyping (SKY), permits the simultaneous visualization of all human chromosomes in 24 different colors. Most chromosomal aberrations detected during cytogenetic evaluation can be resolved using routine cytogenetic techniques alone or in combination with single- or dual-color FISH. However, some cases remain unresolved, in particular de novo supernumerary marker chromosomes and de novo unbalanced structural rearrangements. These findings cause particular diagnostic and counseling concerns when detected during prenatal diagnosis. The purpose of this report is to demonstrate the application of SKY in the characterization of these de novo structural chromosomal abnormalities. Eight cases are described in this report. SKY has considerable diagnostic applications in prenatal diagnosis because of its reliability and speed. The identification of the chromosomal origin of markers and unbalanced translocations provides the patient, physician, and genetic counselor with better predictive information on the phenotype of the carrier.
Subject(s)
Chromosome Aberrations/genetics , Genetic Markers/genetics , Karyotyping/methods , Amniotic Fluid/cytology , Chromosome Banding , Chromosome Disorders , Chromosomes/genetics , DNA Probes/genetics , Fluorescent Dyes/metabolism , Humans , In Situ Hybridization, Fluorescence/methods , Indoles/metabolism , Lymphocytes/cytology , Prenatal Diagnosis/methodsABSTRACT
Expression of liver-enriched trans-acting hepatocyte nuclear factors 1alpha (HNF1alpha) and 4 (HNF4) is correlated with the hepatic phenotype in cultured rat hepatoma cells. We have used a hepatoma variant cell line, H11, that specifically lacks the HNF4 --> HNF1alpha pathway as a model to understand mechanisms controlling hepatic gene expression. We have introduced randomly marked human chromosomes into H11 cells and have isolated a number of microcell hybrids that have rescued hepatic gene expression, including HNF4, HNF1alpha, and alpha1-antitrypsin. Chromosomal analysis of cell hybrids showed that the rescued hepatic phenotype correlated closely with the presence of human chromosome 12p sequences. Although the gene encoding HNF1alpha is located on chromosome 12q24, its retention was not required to rescue the hepatic phenotype. Thus, we suggest that a locus on human chromosome 12p plays an important role in maintenance of hepatic gene expression through activation of the HNF4 --> HNF1alpha pathway.