Transient therapy-related myelodysplastic syndrome associated with monosomy 7 and 11q23 translocation.

Secondary acute myelocytic leukemia (AML) and myelodysplastic syndromes (MDS) are known to develop in patients previously treated with different chemotherapeutic regimens. Nonrandom chromosomal abnormalities have been demonstrated in these therapy-related myeloid disorders which often evolve into refractory AML. The prognosis of these patients with conventional chemotherapy has been dismal and only allogeneic bone marrow transplantation offers a potential cure. We describe two patients who developed MDS after chemo/radiotherapy and had a spontaneous recovery. One patient was treated with MOPP-ABVD hybrid therapy for Hodgkin's disease, developed pancytopenia, marrow hypoplasia and dyserythropoiesis associated with monosomy 7. The other was treated with a combination of chemotherapy including VP-16 for Ewing's sarcoma, developed thrombocytopenia, marrow hypoplasia and dyserythropoiesis associated with an 11q23 translocation. Both patients received rhG-CSF after their cycles of chemotherapy and were considered for a bone marrow transplant. Marrow aspirates at frequent intervals showed gradual disappearance of the abnormal clone with parallel normalization of the peripheral count. In both patients G-CSF might have played a role in the development of the abnormal clone. We suggest that patients with therapy-related MDS without excess of blasts could be closely monitored for karyotypic and hematological improvement rather than transplanted immediately.

Metaphyseal dysostosis and congenital nystagmus in a male infant with the fragile X syndrome.

A 14-month-old male with congenital nystagmus, sparse scalp hair, protuberant ears, developmental delay, and radiologic manifestations of mild metaphyseal dysostosis was coincidentally found to have the fra(X) chromosome in 67% of analyzed metaphases. This observation underscores the need for fra(X) analyses in children with developmental deficit of unknown cause.

Fluorescence in situ hybridization (FISH) of a whole-arm translocation involving chromosomes 18 and 20 with alpha-satellite DNA probes: detection of a centromeric DNA break?

Fluorescence in situ hybridization (FISH) with alpha-satellite DNA probes was used to study whole-arm chromosome translocation products in a family in which the propositus was shown to have a monosomy 18p/trisomy 20p imbalance. By this approach, we show that the chromosome 18 alpha-satellite DNA block is split into 2 smaller units, whereas the chromosome 20 breakpoint is not included within the alpha-satellite DNA region. We found no evidence to suggest that this split alpha-satellite DNA region has reduced or impaired the function of the centromere or that it contributed to the phenotype of the propositus. The FISH technique critically demonstrated the involvement of a whole-arm translocation in this case and provided accurate identification of breakpoints, which was not possible with standard banding techniques.

Mosaic vs. nonmosaic trisomy 9: report of a liveborn infant evaluated by fluorescence in situ hybridization and review of the literature.

We report on a newborn infant with multiple congenital anomalies and apparent nonmosaic trisomy 9 in the blood (by conventional cytogenetic studies) who died shortly after birth. Clinical observations at birth and autopsy are compared with phenotypes of mosaic and nonmosaic trisomy 9 cases reported previously. Unlike the initial cytogenetic analysis, fluorescence in situ hybridization (FISH) studies of metaphase and interphase blood cells and skin fibroblasts detected the presence of euploid and trisomy 9 cells. These results suggest that earlier reports of trisomy 9, which relied on conventional chromosome analysis of a few metaphase cells and/or only one tissue type, may not have excluded mosaicism, and that trisomy 9 may be viable only in the mosaic state.

Angelman syndrome in a daughter with del(15) (q11q13) associated with brachycephaly, hearing loss, enlarged foramen magnum, and ataxia in the mother.

We report on a 4-year-old girl with Angelman syndrome who has an apparent de-novo del(15) (q11q13) originating from a maternally derived chromosome. Her mother had severe brachycephaly, sensorineural hearing loss, speech impediment, and mild ataxia. CT brain scans showed an enlarged foramen magnum in the mother and daughter but magnetic resonance imaging (MRI) showed no brainstem abnormality in either. This family demonstrates that some Angelman syndrome cases may be dominantly transmitted with variable expression and associated with abnormal or cytogenetically apparently normal chromosome 15.

Incidence of 15q deletions in the Angelman syndrome: a survey of twelve affected persons.

Prometaphase chromosome study of 12 persons with an established diagnosis of the Angelman syndrome demonstrated that 5 had a 15q12 deletion appearing similar to that commonly observed in the Prader-Willi syndrome. Phenotype-karyotype correlation did not show any obvious clinical differences between those with and those without the deletion and no clinical overlap between Angelman and Prader-Willi syndrome was apparent. Our survey suggests that 15q12 deletions are frequent in Angelman syndrome but presence of the deletion does not appear to distinguish different clinical phenotypes. Experience with the cytogenetic study of Prader-Willi syndrome predicts that considerable complexity will emerge between the presence of 15 chromosome abnormalities and clinical expression of Angelman syndrome.

Maternal origin of 15q11-13 deletions in Angelman syndrome suggests a role for genomic imprinting.

Six persons with the classical Angelman syndrome (AS) phenotype and de novo deletions of chromosome 15q11-q13 were studied to determine the parental origin of the chromosome deletion. Four of the 6 patients had informative cytogenetic studies and all demonstrated maternal inheritance of the deletion. These findings, together with other reported cases of the origin of the chromosome 15 deletion in AS, suggest that deletion of the maternally contributed chromosome leads to the AS phenotype. This contrasts with the Prader-Willi syndrome (PWS) in which a similar deletion of the paternally contributed chromosome 15 is observed. In deletion cases, a parental gamete effect such as genomic imprinting may be the best model to explain why apparently identical 15q11-q13 deletions may develop the different phenotypes of AS or PWS.

Quantitative calibration and use of DNA probes for investigating chromosome abnormalities in the Prader-Willi syndrome.

Ten genomic DNA probes, subcloned from inserts derived from a phage library constructed from the DNA of flow-sorted chromosomes, have now been mapped to locations within 15q11-15q13. By dosage blotting and densitometry, 5 of these probes map to the 15q11.2-15q12 segment missing in one 15 chromosome of a Prader-Willi syndrome (PWS) patient with a prominent cytological deletion. A sixth probe most likely maps to the same region. The other 4 probes map outside of this segment but within 15q11-15q13. Several of the 15q11.2-15q12 probes, and a cDNA probe homologous to one, have been used to test the DNA from 8 patients exhibiting a wide range of the clinical manifestations expected for PWS patients. DNA deletion was observed in all 3 patients with cytological 15q1 deletions as well as in a patient with an unbalanced (Y;15) translocation. DNA from 1 PWS patient with an unbalanced (5;15) translocation and an inverted duplication of the short arm and proximal long arm of 15 showed at least 1 and possibly 2 extra copies of each genomic probe tested. In the other 3 patients with no cytological deletions, no DNA deletions were found. Thus, the molecular probes described can be used in most PWS patients to analyze the region of proximal 15q implicated in this syndrome.

Recent experience in prenatal fra(X) detection.

At least 35 cases of prenatal fra(X) diagnosis have been confirmed and reported. Amniotic fluid, fetal blood and chorion -ic villus samples have exhibited fra(X) (q27.3) in cultures from 26 males and 9 females. Here we have detected fra(X) in female and male amniotic fluid specimens, AF1/fra(X),X and AF2/fra(X),Y, respectively, and a male CVS/fra(X),Y using both FUdR and excess thymidine (THY) to demonstrate the marker chromosome. Both FUdR and THY detected fra(X) and usually FUdR was superior to THY with the exception of placental cultures. It was important to examine more than one culture per protocol since no fra(X) was observed in one AF2 FUdR culture while another exhibited 19.2% expression. Similarly, confirmation studies in lung fibroblast cultures for AF2 exhibited 4.3% fra(X) in one lab while another found negative results. A similar observation in whole blood cultures was also made recently by us. In addition, we have recently experienced our first false negative fra(X),X prenatal diagnosis. We have observed another case where only one cell in 300 exhibited fra(X) where the male fetus was 50% at-risk and was referred to us after the 20th week of gestation by sonography. On the basis of our experience we recommend the following: 1) the excess THY fra(X) induction system is effective but not superior to FUdR; 2) at least two duplicate cultures per induction system should be analyzed for the marker chromosome to avoid the possibility of false-negative diagnosis; 3) where fra(X) is not demonstrated or is present in very low frequencies in CVS and/or amniotic fluid cultures, complementary DNA marker studies and/or fetal blood cultures must be made available; 4) gestational age dating by ultrasonography is recommended as early as possible.

An atypical Turner syndrome patient with ring X chromosome mosaicism.

Small marker chromosomes (SMC) associated with severe Turner syndrome (TS) variants often represent reduced X chromosomes lacking the X inactivation center (XIC), perturbed dosage compensation, and unbalanced gene expression. A TS patient with mental retardation (MR), unusually short stature, facial and limb malformations, and karyotypic mosaicism involving SMCs is described. Cytogenetic and fluorescence in situ hybridization (FISH) studies of blood and lymphoblastoid cells showed that the SMC was X-chromosome derived, contained a functional centromere, and had ring formation. Karyotypes of 45/46,X,r(X) in blood cells and 45,X/46,-XX/46,X,r(X)/47,X,r(X), + r(X) in fibroblasts were found. Late-replication of the SMC was inconclusive, but the X inactivation specific transcript (XIST) locus within XIC was demonstrated by fluorescent in situ hybridization (FISH). Mechanisms are reviewed that can account for our patient's unusual TS phenotype.

Mosaic trisomy 8: a cautionary note regarding missed antenatal diagnosis.

Chromosomal analysis of fetal cells is a commonly used, safe, and highly accurate procedure. The rate of false-negative results is unknown. Recent experience at four centers suggests that there may be a particular likelihood for mosaic trisomy 8 to be missed with routine antenatal diagnostic procedures. This report reviews these cases, the characteristic findings of mosaic trisomy 8, and the tissue-specific differential yield of chromosomal analysis that may contribute to the increased risk of missed antenatal diagnosis in patients with this disorder.

Cytogenetic survey for autistic fragile X carriers in a mental retardation center.

A cytogenetic survey of 67 individuals previously identified as having mental retardation and autistic behaviors revealed 1 person (1.5%) with the fragile X chromosome (fra[X]) and 3 (4.5%) with autosome abnormalities. This low prevalence of fra(X) indicates that most persons with fra(X) in this mental retardation center did not have autistic behaviors severe enough to be identified as a secondary psychiatric diagnosis. The presence of other chromosomal abnormalities is consistent with the known causal heterogeneity of autism in mental retardation populations.

Fragile (X) expression: relationship to the cell cycle.

The fragile (X) chromosome demonstrable in individuals with one type of X-linked mental retardation is seldom, if ever, seen in more than 50% of cells of affected individuals. We have devised a model to explain this apparent 50% maximum, one essential feature of which is that the fragile (X) will not be seen in cells in their first division in thymidine-depleted media. The validity of our model was tested on lymphoblastoid cell lines from affected males by treating the cells with fluorodeoxyuridine (FUdR) to induce the marker and/or bromodeoxyuridine (BrdU) to determine the cell cycle. We have evidence that the fragile (X) is present in cells in the first and subsequent cell divisions in thymidine-depleted media. In light of these observations our model is not valid and the 50% expression of the fragile site at Xq(28) and other unusual properties of this region of the X chromosome remain unexplained.

Localization of chromosomal DNA sequences homologous to ribosomal gene type I insertion DNA in Drosophila melanogaster.

Chromosomal sites which have DNA homology to the 1 kb (kilobase pair) BamHI restrictable fragment of the 5 kb type I insertion present in many ribosomal genes in Drosophila melanogaster, were identified by using in situ hybridization and autoradiography. XX and XY complements of polytene chromosomes showed the nucleolus and chromocenter to be heavily labeled. Of the light label over euchromatic regions, the 102C band of chromosome 4 labeled particularly intensely. In mitotic XX and XY complements, the NORs (nucleolus organizer regions) of both sex chromosomes labeled as did the centromeric heterochromatin of autosomes. Label also appeared less frequently over telomeric and euchromatic regions.

Unusual cytogenetic mosaicism involving chromosome 14 abnormalities in a child with an MR/MCA syndrome and abnormal pigmentation.

An 8-year-old female child with mental retardation (MR), multiple congenital anomalies (MCA) and irregular pigmentation was shown to have karyotypic mosaicism involving chromosome 14 abnormalities. Four cell lines were found in both peripheral blood lymphocytes and skin fibroblasts and were represented by: a normal karyotype, an isopseudodicentric 14q [iso psu dic(14)], a ring 14 [r(14)], and a monosomy 14 [mono(14)]. Our results are compared with reported cases involving multiple abnormalities of specific chromosomes. Karyotypic mosaicism of comparable chromosome 14 abnormalities is rare, with only one known previous case. Detailed analysis of karyotypic mosaicism of rare chromosomal abnormalities is essential to determine meaningful correlations with specific patterns of malformation.

Paternal uniparental disomy of chromosome 15 in a child with Angelman syndrome.

Angelman and Prader-Willi syndromes are clinically distinct neurobehavioral disorders most commonly resulting from large deletions of chromosome 15q11-q13. The deletions arise differentially during maternal or paternal gametogenesis, respectively. A subgroup of patients with either syndrome have no apparent deletion, and because many such patients with Prader-Willi syndrome display inheritance of two copies of chromosome 15 from the mother only (uniparental disomy; UPD), we suggested that paternal UPD might be found in patients with Angelman syndrome. We report here clinical, cytogenetic, and molecular evidence on the 1 patient with paternal UPD for chromosome 15 who was found in our study population. This represents, to our knowledge, the first patient with paternal UPD to be studied with DNA probes from the chromosome 15q11-q13 critical region. In contrast to our findings for patients with Prader-Willi syndrome, in which maternal UPD was common, our data demonstrate that paternal UPD is infrequent in patients with Angelman syndrome.

Fragile (X) expression induced by FUdR is transient and inversely related to levels of thymidylate synthase activity.

Thymidylate synthase (TS) activity was monitored in fluorodeoxyuridine (FUdR)-treated lymphoblasts from individuals carrying the fragile (X) [fra(X)] chromosome. Fra(X) expression and levels of TS activity were measured over a 72-hr period at different cell densities. TS activity was 80%-90% inhibited immediately after exposure to FUdR and remained suppressed for the first 24 hrs. Fra(X) expression was not found until 6-8 hrs after FUdR treatment, and at 24 hrs, reached a maximum expression of approximately 50%. At 48 and 72 hrs, however, increasing levels of TS activity paralleled a dramatic drop in fra(X) expression. High fra(X) expression at 48 and 72 hrs could be maintained by rechallenging cultures with increasing doses of FUdR. At low cell densities, fra(X) expression was maintained at high levels for a much longer period of time. In two lymphoblastoid cell lines from obligate carriers, which either expressed at very low levels or did not express the fra(X) in routine cultures, TS activity was also 90% inhibited but with no corresponding fra(X) expression 12 or 24 hrs after FUdR treatment. We conclude that: FUdR inhibits TS activity immediately and induces fra(X) expression 6-8 hrs later, FUdR-induced fra(X) expression and TS activity are inversely related, the FUdR effect on fra(X) expression and TS activity is time and cell-density dependent, and inhibition of TS activity is a necessary but not sufficient condition for fra(X) expression.

Selective protection of specific DNA sequences in the heterochromatin of C-banded human Y chromosomes.

The molecular basis of C-banding was investigated by in situ hybridization of human Y chromosome-derived repeated sequences, DYZ1 and DYZ2, to untreated or to alkaline-treated metaphases. Autoradiography of G-banded metaphases showed that both probes hybridized to the long arm of Y. Alkaline hydrolysis significantly reduced grain number for DYZ2 (58%-82%; P less than .05) but not for DYZ1 (P greater than .05). Similar results were observed for interphase nuclei. These findings demonstrated that the heterochromatin of the long arm contains at least two repetitive DNA fractions having two different sensitivities to alkaline hydrolysis. These observations support the notion that DYZ2 maps terminally on the Yq arm and may be nonheterochromatic.

Xp pseudoautosomal gene haploinsufficiency and linear growth deficiency in three girls with chromosome Xp22;Yq11 translocation.

Colony stimulating factor-2 receptor alpha (CSF2RA) and interleukin-3 receptor alpha (IL3RA), two genes from the chromosome Xp and Yp pseudoautosomal region (PAR), have been suggested as candidate genes for short stature in Turner syndrome. We report three girls with X;Y translocation (46,X,der(X)t(X;Y)(p22;q11) initially detected by amniocentesis. The terminal portion of the X chromosome distal to the translocation breakpoint at Xp22 was deleted on the derivative X chromosome in all three patients. Each had normal stature at birth, with greater than expected deceleration of growth velocity by the second year. Using fluorescence in situ hybridisation (FISH), we have shown deletion of the CSF2RA and IL3RA loci on the derivative X chromosomes of all three patients. The role of CSF2RA and IL3RA haploinsufficiency in linear growth and final adult stature is discussed. Additional studies, particularly of molecular deletions within the PAR, are needed to improve our understanding of the role of these and other PAR loci in the genetic control of adult stature.

Association of pigmentary anomalies with chromosomal and genetic mosaicism and chimerism.

We have evaluated eight patients with pigmentary anomalies reminiscent of incontinentia pigmenti or hypomelanosis of Ito. All demonstrated abnormal lymphocyte karyotypes with chromosomal mosaicism in lymphocytes and/or skin fibroblasts. In seven the skin was darkly pigmented, and in all of these seven cases the abnormal pigmentation followed Blaschko lines. The literature contains at least 36 similar examples of an association between pigmentary anomalies and chromosomal mosaicism, as well as five examples of an association with chimerism. The pigmentary anomalies are pleomorphic, and the chromosomal anomalies involve autosomes and sex chromosomes. The pigmentation patterns are reminiscent of the archetypal paradigm seen in allophenic mice and demonstrate the clonal origin of melanoblasts from neural crest precursors. Patients with anomalous skin pigmentation, particularly when it follows a pattern of Blaschko lines, should be appropriately evaluated for a possible association with chromosomal or genetic mosaicism or chimerism.