Array comparative genomic hybridization profiling of first-trimester spontaneous abortions that fail to grow in vitro.

OBJECTIVES: Cytogenetic analysis of spontaneous abortion samples can be limited by culture failure. Failure to grow in vitro has traditionally been suspected to be due to in vivo death of tissue associated with spontaneous abortion (SAB) or simply technical factors of growth in culture. METHOD: We used array comparative genomic hybridization (array CGH) to investigate chromosomal imbalances in products of conception that failed to grow in vitro. RESULTS: Our data on 26 cases of SABs that failed to grow in culture are compared and contrasted with published data on cytogenetic findings following in vitro culture. The results revealed abnormalities uncommonly seen by classic cytogenetic methods. These abnormalities include high rates of double aneuploidy and autosomal monosomy. The data taken together suggest that classic cytogenetics of spontaneous abortion may yield normal karyotypes or selected abnormal karyotypes that permit cell proliferation in vitro while Array CGH detects other abnormalities. CONCLUSION: Array CGH is becoming an important clinical assay for unbalanced chromosome abnormalities whether cells grow in culture or not and in cases of analysis on one or few cells.

Morphological and cytogenetic analysis of intact oocytes and blocked zygotes.

We examined cytological and cytogenetic parameters of 1076 oocytes and 385 zygotes that failed to develop post in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI). Out of 1076 oocytes, 894 (83%) arrested oocytes showed a first polar body and were thus assumed arrested at metaphase II while the remainder showed no polar body. In the group of oocytes with a polar body, 20.5% had an abnormal karyotype. Cytologically, premature sperm chromosome condensation was noted in 28.3% of uncleaved oocytes. This high PCC can be explained by the different grades of oocyte maturity from one center to another. Oocytes from older women showed no increased aneuploidy but did show increased premature chromosome condensation. Analysis by classical technique of 220 uncleaved zygotes showed 91 with highly condensed chromosomes, 53 with asynchrony of condensation, 31 with pulverized chromosomes, and 45 arrested at the first somatic metaphase. Out of 385 arrested zygotes, 165 were explored by in situ hybridization. FISH using a set of 7 chromosome-specific probes showed aneuploidy in the chromosomes analyzed (13, 16, 18, 21, 22, X, Y) in 21.8% of blocked zygotes (19-25% depending on morphology). Extrapolating to other chromosomes, we expect that a vast majority of blocked zygotes and oocytes probably carry chromosome abnormalities. These data demonstrate the contributions of chromosome disorder in early embryo development blocking and implantation failure. Certainly, the issue of cytoplasm and nuclear immaturity and their relation to each other and to chromosome abnormalities provides a fertile area for future investigation in ART.

ETV6/CBFA2 fusions in childhood B-cell precursor acute lymphoblastic leukemia with myeloid markers.

A translocation resulting in a fusion of ETV6 (TEL) gene at 12p13 and CBFA2 (AML1) gene at 21q22 is variably reported in 16-36% of cases of childhood acute lymphoblastic leukemia (ALL). This t(12;21)(p13;q22) is not detectable by conventional cytogenetic methods and was reported to be associated with B-cell precursor ALL with presumed favorable prognosis. We have examined 18 cases of well characterized childhood B-cell precursor ALL with cytogenetic, immunophenotypic, and clinical data for the presence of the t(12;21) using fluorescence in situ hybridization (FISH). Fourteen of the 18 cases (78%) were positive for fusion ETV6/CBFA2. One of seven adult ALL patients was positive (12% of cells positive in this 21 year old patient). By contrast, no evidence of t(12;21) by FISH was noted in two childhood T-ALL cases and 10 normal bone marrow samples. Twelve of the 14 positive childhood cases had CD13 and/or CD33 expression (myeloid markers) while only one of the four negative cases was CD13 and CD33 positive. Eight of 12 cases positive for t(12;21), and with conventional cytogenetic data, had structural and/or numerical chromosome abnormalities other than the detected t(12;21). One case had relapse with gradual increase in percentage of cells positive for t(12;21) and development of an isochromosome 21 carrying the fusion signals. The data reveal a strong association of t(12;21) with B-cell precursor ALL, especially with myeloid marker expression.

Increased incidence of trisomy 8 in acute myeloid leukemia with skin infiltration (leukemia cutis).

Anecdotal literature reports and the authors' own observations suggest an association between chromosome 8 aneuploidy and leukemia cutis. The authors investigated this potential association by using fluorescence in situ hybridization (FISH) directly on skin infiltrates in a series of 11 patients with acute monocytic leukemia (AML). Seven of the 11 patients were aneuploid for chromosome 8 by FISH which was confirmed by dual color hybridization. Six of these seven patients were AML-M4 or M5 and one was M1. The majority of the cases with leukemia cutis expressed CD4 (90% of cases), CD14 (60%), and/or CD56 (50%) in bone marrow leukemic cells. The data show the utility of examination of skin infiltrates by FISH for the detection of trisomy 8 in leukemia cutis. They also suggest the importance of trisomy 8 as a factor in predisposition to skin infiltration in AML.

Three probands with autistic disorder and isodicentric chromosome 15.

We have identified three unrelated probands with autistic disorder (AD) and isodicentric chromosomes that encompass the proximal region of 15q11.2. All three probands met the Diagnostic and Statistical Manual of Mental Disorders, fourth edition [DSM-IV; American Psychiatric Association, 1994], and International Classification of Diseases ( ICD-10) diagnostic criteria for AD, confirmed with the Autism Diagnostic Interview -Revised (ADI-R). Chromosome analysis revealed the following karyotypes: 47,XX,+idic(15)(q11.2), 47,XX, +idic(15) (q11.2), and 47,XY,+idic(15)(q11.2). Haplotype analysis of genotypic maker data in the probands and their parents showed that marker chromosomes in all three instances were of maternal origin. Comparison of the clinical findings of the three AD probands with case reports in the published literature (N = 20) reveals a clustering of physical and developmental features. Specifically, these three probands and the majority of reported probands in the literature exhibited hypotonia (n = 13), seizures (n = 13), and delayed gross motor development (n = 13). In addition, clustering of the following clinical signs was seen with respect to exhibited speech delay (n = 13), lack of social reciprocity (n = 11), and stereotyped behaviors (n = 12). Collectively, these data provide further evidence for the involvement of chromosome 15 in AD as well as present preliminary data suggesting a clustering of clinical features in AD probands with proximal 15q anomalies.

Cytogenetics and mechanisms of spontaneous abortions: increased apoptosis and decreased cell proliferation in chromosomally abnormal villi.

Genetic defects of the zygote, such as chromosome aberrations, are the most frequent causes of abnormal embryonic development and spontaneous abortion. However, the underlying mechanisms remain unknown. Chromosome aberrations likely cause changes in placental morphology and function (such as size, shape, vascularity, and the presence of trophoblastic inclusion). We postulated that chromosome aberrations may affect rates of cell proliferation or programmed cell death (apoptosis) during the differentiation of chorionic villi. To address these questions, we evaluated cell proliferation using a monoclonal antibody to Ki-67 (a cell-cycle marker) and apoptosis using the in situ end-labeling method (TUNEL) on paraffin-embedded placental tissues. Tissues were obtained from spontaneous abortions in early gestational periods with normal (11 cases) and abnormal karyotypes (15 cases), as well as eight normal control placentas from elective abortions. Apoptotic cells were found in the stroma of all cases, but were significantly higher in number in the stroma of chromosomally abnormal versus chromosomally normal spontaneous abortions. The apoptotic index of the trophoblasts was not significantly different between groups. Cell proliferation was higher in muscularized blood vessels in chromosomally normal placentas (both elective and spontaneous abortions) versus chromosomally abnormal spontaneous abortions. Cell proliferation was different in the trophoblast and stroma between the groups but to a lesser degree than in blood vessels. The morphological and biological data presented here suggest that: (1) chromosomally abnormal spontaneous abortions may occur because of different mechanisms than chromosomally normal spontaneous abortions, (2) apoptosis of the stromal cells and cell proliferation in blood vessels and stroma play an important role in the differentiation and functioning of villi, and (3) these changes could explain the etiology of spontaneous abortion and growth retardation of chromosomally abnormal embryos.

Comparative genomic hybridization studies in hydatidiform moles and choriocarcinoma: amplification of 7q21-q31 and loss of 8p12-p21 in choriocarcinoma.

Comparative genomic hybridization (CGH) was utilized to investigate genetic changes from archived cases of choriocarcinoma (n = 12) and hydatidiform moles (n = 7). Test DNA was extracted from paraffin-embedded tissues, amplified using total universal PCR, and co-hybridized with control DNA to normal metaphases. Comparative genomic hybridization findings showed chromosomal imbalances in 9 of 12 cases of choriocarcinoma. By contrast, all hydatidiform moles showed normal CGH profiles. Consistent findings in choriocarcinoma included deletion at 8p (5 cases) and amplification at 7q (4 cases). A tumor suppressor gene (e.g., N33) at 8p and/or a growth regulator at 7q could play a role in the initiation of choriocarcinoma and its progression. This is the first study showing specific alterations in choriocarcinomas by CGH, and illustrates the utility of this technique in elucidating genetic changes in gynecological tumors.

Tetrasomy 8 evolving into a segmental triplication 8q in a case of acute monocytic leukemia.

We report a case of AML-M5 with tetrasomy 8 that evolved within a 7-month period to a segmental triplication 8q. Other numerical abnormalities in the initial diagnosis were not found at the relapse; however, a chromosome 1 structural abnormality was maintained, proving the clonal evolution from tetrasomy 8 to a segmental triplication of the long arm of 8. This strongly suggests that there is a functional and selective advantage for duplications and triplications of 8q in these patients.

Induction of DNA replication in adult rat neurons by deregulation of the retinoblastoma/E2F G1 cell cycle pathway.

In adult organisms, a range of proliferative capacities are exhibited by different cell types. Stem cell populations in many tissues readily enter the cell cycle when presented with serum growth factors or other proliferative cues, whereas "terminally" postmitotic cells, such as cardiac myocytes and neurons, fail to do so. Although they rarely show evidence of a proliferative capacity in vivo, there is accumulating evidence to suggest that DNA synthesis can be triggered in postmitotic cells. We now show that cultured adult rat sensory neurons can replicate DNA in response to ectopic expression of E2F1 or E2F2 and that this is augmented by expression of cyclin-dependent kinase activities. We also find that addition of serum and laminin inhibits the E2F-induced S-phase in neurons but not in nonneuronal cells in the same cultures. We conclude that, although terminally differentiated neurons possess the capacity to reinitiate DNA replication in response to G1 regulatory activities, they fail to do so in the presence of signals that do not inhibit S-phase in other cell types in the same cultures. This suggests the existence of cell type-specific inhibitory pathways induced by these signals.

Genetic studies of autistic disorder and chromosome 7.

Genome-wide scans have suggested that a locus on 7q is involved in the etiology of autistic disorder (AD). We have identified an AD family in which three sibs inherited from their mother a paracentric inversion in the chromosome 7 candidate region (inv(7)(q22-q31.2)). Clinically, the two male sibs have AD, while the female sib has expressive language disorder. The mother carries the inversion, but does not express AD. Haplotype data on the family suggest that the chromosomal origin of the inversion was from the children's maternal grandfather. Based on these data, we have genotyped 76 multiplex (>/=2 AD affecteds/family) families for markers in this region of 7q. Two-point linkage analysis yielded a maximum heterogeneity lod score of 1.47 and maximum lod score (MLS) of 1.03 at D7S495. Multipoint MLS and NPL analyses resulted in peak scores of 1.77 at D7S2527 and 2.01 at D7S640. Examination of affected sibpairs revealed significant paternal (P = 0.007), but not maternal (P = 0. 75), identity-by-descent sharing at D7S640. Significant linkage disequilibrium was detected with paternal (P = 0.02), but not maternal (P = 0.15), transmissions at D7S1824 in multiplex and singleton families. There was also evidence for an increase in recombination in the region (D7S1817 to D7S1824) in the AD families versus non-AD families (P = 0.03, sex-averaged; and P = 0.01, sex-specific). These results provide further evidence for the presence of an AD locus on chromosome 7q, as well as provide evidence suggesting that this locus may be paternally expressed.

De novo highly complex chromosome rearrangement (CCR) involving five breakpoints with congenital anomalies analyzed by FISH.

We report on a child with ptosis, epicanthal folds, depressed nasal bridge, carp-shaped mouth, low set ears, hirsutism, pectus excavatum, and developmental and language delay presenting with a balanced complex chromosomal rearrangement (CCR). R- and G-banding methods and fluorescence in situ hybridization were used to document that this is a complex translocation with five breakpoints involving chromosomes 1, 7, 10 and 21.

Structure and function of the nucleus: anatomy and physiology of chromatin.

A review of the literature accumulated recently on nuclear structure and function reveals that: (1) The nucleus is the interphase form of chromosomes (chromatin organizes and compartmentalizes the nucleus). (2) These organizational programs are morphogenetic in nature and are regulated by both DNA content and by epigenetic interactions. (3) In mammals with a diploid complement, it is very likely that chromosomes construct interphase domains based on their structural milieu (including any imprinted areas). These are the same structured areas that correspond to G- and R-bands with their varying DNA content and early versus late replication. (4) Changes in a position of a segment of DNA from one chromatin environment to another changes its availability to early replication factors and transcription factors as well as its nuclear positioning and chromatin architecture. This process was first described as positional effect variegation in Drosophila but is now found to be more general and explains many cases of direct clinical relevance. Examples in mammals include spreading of X inactivation, imprinting and changes in chromatin associated with chromosome translocation. (5) Chromosomal autoconstruction and reconstruction into a functional nucleus are altered during cell cycle and during differentiation (much more work needed on this area).

Translocation (15;17)(q22;q21) as a secondary chromosomal abnormality in a case of acute monoblastic leukemia with tetrasomy 8.

We describe a case of acute monoblastic leukemia (AML M5a), originally presenting as granulocytic sarcoma of the testis, showing unusual cytogenetic abnormalities. Tetrasomy 8 (primary) and t(15;17)(q22;q21) (secondary) were detected in bone marrow cells 6 months post-diagnosis, both by routine karyotype analysis and by fluorescence in situ hybridization (FISH) studies on metaphases and interphase nuclei. Retrospectively, the same abnormalities were identified in the primary testicular lesion using interphase FISH. However, reverse transcriptase polymerase chain reaction (RT-PCR) did not reveal the presence of a classic PML/RAR alpha fusion transcript. To the best of our knowledge, this is the first case to be reported in the literature of AML showing tetrasomy 8 in combination with secondary t(15;17).

A potential model for early stages of chromosomal evolution via concentric Robertsonian fans: a large area of polymorphism in southern short-tailed shrews (Blarina carolinensis).

Western Tennessee contains unusually highly polymorphic populations of southern short-tailed shrews (Blarina carolinensis). We previously documented eight Robertsonian translocations (ROBs) accounting for a variation in diploid number from 46 in most of this species' range to 34-40 in western Tennessee. We have now expanded our study to include data from adjacent areas in Tennessee and Mississippi, 10 localities in all. The new data show a variation in diploid number ranging from 31 to 41, four new ROBs (for a total of 12), and the novel finding of monobrachial translocations in this group. All animals collected from this large area (extending over 12, 000 km(2)) had some level of ROBs, and none represented the 2n = 46 form seen in other parts of the range of this species. Because other species of shrews (genus Sorex) are not affected in the same area, the factors and/or selective forces causing this extensive polymorphism in B. carolinensis must be unique to this species and to this geographic area. Some ROBs were found throughout this large area of over 12,000 km(2). Other translocations (including those with monobrachial homology) were located in one or two localities in this large area, and still other translocations were intermediate in their distribution. There was a concentric pattern to the evolution and presumed spreading of the ROBs. This allowed us to expand the concept of a Robertsonian "fan," introduced by Matthey (1970), to that of concentric evolution of multiple fusion fans: ROBs likely arose independently, separated temporally and geographically, and radiated into surrounding populations to create this complex zone of polymorphism. This is an active process in its infancy, and it is not as mature as that seen in European studies of Mus and Sorex.

A novel chromosomal rearrangement associated with therapy-related acute leukemia.

We describe a 7-year-old girl with therapy-related acute myeloid leukemia (AML) associated with a single and novel karyotypic abnormality. The patient had been treated with alkylating agents and etoposide for hypothalamic pilocytic astrocytoma at age 17 months, and developed mixed lineage AML. Cytogenetic analysis of the leukemic blasts showed 46,XX,der(7)t(7;11)(q22;q14) in all cells examined. Southern blot analysis revealed three copies of an unrearranged MLL gene on chromosome 11q. This is the first report of a triplicated, unrearranged MLL gene in association with a deletion of 7q anomaly and an unbalanced translocation in therapy-related leukemia.

Unconjugated estriol as an indication for prenatal diagnosis of steroid sulfatase deficiency by in situ hybridization.

BACKGROUND: Undetectable or very low unconjugated estriol (E3) levels in routine maternal serum screening are associated with steroid sulfatase deficiency, miscarriages, and anencephaly. CASES: Fluorescence in situ hybridization techniques were used in the diagnosis of steroid sulfatase deficiency prenatally in three cases with low or undetectable unconjugated E3 levels. Results showed a male fetus with a deleted steroid sulfatase region, but intact Kallmann syndrome region in all three cases. One mother was studied by fluorescence in situ hybridization and showed a similar deletion for steroid sulfatase gene in one copy of X chromosome (carrier). CONCLUSION: Women with undetectable or very low levels of estriol on serum screening should be counseled regarding steroid sulfatase deficiency with evaluation by fluorescence in situ hybridization.

Chromosome abnormalities in the placenta and spontaneous abortions.

Data are presented confirming that placental chromosome abnormalities are more important than fetal chromosome abnormalities in determining fetal loss. Improved methodologies for studying chromosome abnormalities in spontaneous abortion (SAB) are presented that include results for 141 cases (gestational ages 6-24 weeks) with karyotypic study on placental as well as fetal tissue. Experience in two laboratories gave a success rate of >90% of specimens with identifiable placental tissues, an average turnaround time of 10 days for those that produced chromosome results, male-to-female ratio of >50% (indicating no impact of maternal cell contamination), and 30% of cases had chromosome abnormalities. More significantly, two cases (5% of all abnormal cases) showed an abnormal karyotype limited to the placenta. This illustrates the need to examine the placenta in cases of SAB and the importance of technical issues in laboratory studies.

No requirement for V(D)J recombination in p53-deficient thymic lymphoma.

The p53 tumor suppressor is activated in response to a variety of cellular stress signals, although specific in vivo signals that trigger tumor suppression are unknown. In mouse thymocytes, where p53 inactivation leads to tumorigenesis, several observations suggest that V(D)J recombination of T-cell receptor (TCR) loci could provide a DNA damage signal triggering p53-dependent apoptosis and tumor suppression. Inactivation of p53 would allow V(D)J driven mutation of additional cancer genes, facilitating tumorigenesis. Here, we show that mice with a p53 deficiency in thymocytes and unable to carry out V(D)J recombination are not impaired in the development of thymoma. Recombination-activating gene (RAG) deficiencies were introduced into both p53-/- mice and TgTDeltaN transgenic mice, a strain in which 100% of the mice develop thymoma due to thymocyte-specific inactivation of p53 by a simian virus 40 T-antigen variant. V(D)J recombination was dispensable for tumorigenesis since thymomas developed with or without the RAG-1 or RAG-2 gene, although some delay was observed. When V(D)J recombination was suppressed by expression of rearranged TCR transgenes, 100% of the TgTDeltaN mice developed thymoma, surprisingly with reduced latency. Further introduction of a RAG deficiency into these mice had no impact on the timing or frequency of tumorigenesis. Finally, karyotype and chromosome painting analyses showed no evidence for TCR gene translocations in p53-deficient thymomas, although abundant aneuploidy involving frequent duplication of certain chromosomes was present. Thus, contrary to the current hypothesis, these studies indicate that signals other than V(D)J recombination promote p53 tumor suppression in thymocytes and that the mechanism of tumorigenesis is distinct from TCR translocation oncogene activation.

Clinical characteristics of patients with de novo acute myeloid leukaemia and isolated trisomy 11: a Cancer and Leukemia Group B study.

Isolated trisomy 11 is the third most common sole trisomy in de novo acute myeloid leukaemia (AML). However, only 49 cases have been published, and for only a fraction of these cases has full description of clinical and haematological features been provided. As a result, little is known about the clinical characteristics of de novo AML patients with solitary trisomy 11. We have identified 13 patients (0.9%) with isolated trisomy 11 among a total of 1496 consecutive adult patients successfully karyotyped as part of a prospective Cancer and Leukemia Group B (CALGB) cytogenetic study (CALGB 8461). Nine patients (69%) were over the age of 60 (range 29-73 years). Eight patients (62%) were diagnosed with AML of FAB M2 subtype, three patients (23%) had FAB M1 AML and one patient each had AML of FAB M0 and M7, respectively. Seven patients (54%) had high, >100 x 10(9)/l, platelet counts (median 102 x 10(9)/l; range 17-207 x 10(9)/l). All patients received CALGB induction therapy with standard doses of cytarabine and daunorubicin. Six patients (46%) achieved a complete remission (CR). The median CR duration was 17.5 months (range 8.7-49.8). Only one patient, who underwent bone marrow transplantation in first CR, continues in initial CR. The median survival was 14.3 months (range 0.5-50.7); only one patient survives. We conclude that de novo AML with isolated trisomy 11 is predominantly associated with older age, M2 and M1 FAB subtypes, high platelet count and few long-term disease-free survivals, although it is currently unknown whether isolated trisomy 11 constitutes an independent prognostic factor.