Duplication of the short arm of chromosome 9. Analysis of five cases

Summary Five females with duplication of the short arm of one chromosome 9 are reported, one tetrasomic and four trisomic for 9p. The tetrasomy is due to an isochromosome 9p while the trisomies are due in one case to an intrachromosomal duplication present in lymphocytes but not in fibroblasts, two are secondary to translocations with chromosomes 22 and 13 respectively, and one is a mosaic with a cell line with an additional deleted chromosome 9 present in lymphocytes and fibroblasts. This analysis indicates that duplications 9p may result in impairment of ovarian function. The phenotypic differences between trisomy and tetrasomy 9p are discussed.     

Characterization of a neocentric supernumerary marker chromosome originating from the Xp distal region by FISH, CENP-C staining, and array CGH

A small supernumerary marker chromosome (SMC) was observed in a girl with severe developmental delay. Her dysmorphism included prominent forehead, hypertelorism, down-slanting palpebral fissures, low-set/large ears, and flat nasal bridge with anteverted nares. This case also presented hypotonia, hypermobility of joints, congenital heart defect, umbilical hernia, failure to thrive, and seizures. The SMC originated from the distal region of Xp as identified by FISH with multiple DNA probes. Staining with antibodies to Centromere Protein C (CENP-C) demonstrated a neocentromere, while FISH with an alpha-satellite DNA probe showed no hybridization to the SMC. A karyotype was described as 47,XX,+neo(X)(pter-->p22.31::p22.31-->pter), indicating a partial tetrasomy of Xp22.31-->pter. This karyotype represents a functional trisomy for Xp22.31-->pter and a functional tetrasomy for the pseudoautosomal region given that there is no X-inactivation center in the marker chromosome. The SMC was further characterized by microarray-based comparative genomic hybridization (array CGH) as a duplicated DNA fragment of approximately 13 megabase pairs containing about 100 genes. We have described here a new neocentromere with discussion of its clinical significance.     

RET rearrangements in papillary thyroid carcinomas and adenomas detected by interphase FISH

Activation of the RET protooncogene through somatic rearrangements represents the most common genetic alteration in papillary thyroid carcinoma (PTC). Three main rearranged forms of RET have been described: RET/PTC1 and RET/PTC3, which arise from a paracentric inversion of the long arm of chromosome 10, and RET/PTC2, which originates from a 10;17 translocation. We have developed a dual-color FISH approach to detect RET/PTC rearrangements in interphase nuclei of thyroid lesions. By using a pool of three cosmids encompassing the RET chromosome region and a chromosome 10 centromeric probe, we could discriminate between the presence of an inversion (RET/PTC1 and RET/PTC3) or a translocation (RET/PTC2). We have investigated a series of thyroid tissue samples from Italian and French patients corresponding to a total of 69 PTCs and 22 benign lesions. Among PTCs, 13 (18.8%) showed a RET rearrangement, and 11 (15.9%) of these carried an inversion (RET/PTC1 or RET/PTC3) in more than 10% of the nuclei examined. Activated forms of RET were also observed in three adenomas. RT-PCR analysis on the same samples confirmed the presence and the type of rearrangement predicted using FISH analysis. An interesting difference in the frequency and type of RET rearrangements was detected between the Italian and the French patients. Furthermore, we identified a putative novel type of rearrangement in at least one PTC sample. Several PTCs carried a significant number of cells characterized by a trisomy or a tetrasomy of chromosome 10. Overall, the FISH approach in interphase nuclei represents a powerful tool for detecting, at the single cell level, RET/PTC rearrangements and other anomalies involving the RET chromosome region.     

Chimerism and multiple numerical chromosome imbalances in a spontaneously aborted fetus

We report on a case of chimerism and multiple abnormalities of chromosomes 21, Xand Yin spontaneous abortion specimen. To the best our knowledge the present case is the first documented chimera in a spontaneously aborted fetus. The application of interphase fluorescence in situ hybridization (FISH) using chromosome enumeration and site-specific DNA probes showed trisomy X in 92 nuclei (23 %), tetrasomy X in 100 nuclei (25 %), pentasomy of chromosome X in 40 nuclei (10 %), XXY in 36 nuclei (9 %), XXXXXXYY in 12 nuclei (3 %), XXXXXYYYYY in 8 nuclei (2 %), trisomy 21 and female chromosome complement in 40 nuclei (10 %), normal female chromosome complement in 72 nuclei (18 %) out of 400 nuclei scored. Our experience indicates that the frequency of chimerism coupled with multiple chromosome abnormalities should be no less than 1 : 400 among spontaneous abortions. The difficulties of chimerism identification in fetal tissues are discussed.     

Comparative FISH analysis of numerical chromosome 7 abnormalities in 5-μm and 15-μm paraffin-embedded tissue sections from prostatic carcinoma

 Interphase fluorescence in situ hybridization (FISH) was performed on 15-μm-thick paraffin sections from prostatic carcinomas using a chromosome 7-specific α-satellite DNA probe. A confocal laser scanning microscope (CLSM) was used for optical sectioning of the thick sections and reconstruction of 3D images. The number of FISH signals was determined by a gallery of optical sections evaluating only complete nuclei. To investiate the influence of section thickness and truncation and nuclei on scoring results, we compared the FISH data from 15-μm sections with signal counts obtained from 5-μm sections. The latter were evaluated by conventional fluorescence microscopy in the same tumor regions previously defined and marked on the slides. After statistical analysis of spot frequencies in tumor and non-tumorous cells (χ² test), we transferred the signal frequencies into a cytogenetic classification (−7, +7, polysomy 7). Based on this classification, most cases showed more than one chromosome 7 aberration type. Trisomy 7 (+7) became apparent in 15-μm-thick sections in all 19 tumors, polysomy 7 (>3 spots) in 18/19 cases, and monosomy 7 (−7) in 13/19 cases. In 5-μm sections, however, trisomy 7 and polysomy 7 were found in only 7/19 and 13/19 cases, respectively, and monosomy 7 in 7/19 cases. When comparing the classification results of tumor cells of the same tumor regions originating either from 5-μm or 15-μm sections, the following discrepancies were noted: in 15-μm sections exclusively, in 12/19 tumors, trisomy 7 was found; in 6/19 cases, polysomy 7; in 8/19 cases, monosomy 7. The high proportion of cases with tumor nuclei expressing only one hybridization signal of chromosome 7 in 15-μm sections could be confirmed as monosomy 7 in five selected cases by double-hybridization using centromere-specific probes for chromosomes 7 and 12. These results demonstrate that numerical chromosome 7 aberrations are more frequently observed in thick (15-μm) paraffin-embedded tissue sections by evaluating only complete nuclei. The use of routine sections (5-μm) for interphase cytogenetic analyses is compromised by a remarkable underestimation of the real chromosome copy numbers. Accepted: 7 November 1996     

Molecular cytogenetic identification and characterization of a de novo supernumerary neocentromeric derivative chromosome 13

We report a young girl with microphthalmia, conductive deafness, aortic isthmus stenosis, laryngomalacia, and laryngeal stenosis carrying a de novo supernumerary neocentromeric derivative chromosome 13. For the precise identification and characterization of the eu- and heterochromatic content of the marker chromosome, straightforward molecular cytogenetic analyses were performed, such as chromosome microdissection, FISH with different probes (e.g. wcp, alphoid centromeric probes, BAC), centromere-specific multicolor FISH (cenM-FISH), and multicolor banding (MCB). The analyses demonstrated that the marker consisted of an inverted duplication (partial tetrasomy) of the distal portion of chromosome 13 that was separated from the endogenous chromosome 13 centromere. Using an all-centromere probe and multicolor cenM-FISH, no alpha-satellite DNA hybridization signal was detectable on any portion of the derivative chromosome. The presence of a functional and active neocentromere on the derivative chromosome 13 was confirmed by positive immunofluorescence signals with CENP-C antibodies. BAC-FISH confirmed the cytogenetic localization of the neocentromere in band 13q31.3. Thus the patient had a mosaic conventional karyotype mos 47,XX,+inv dup(13)(qter-->q21.3::q21.3-->q31.3-->neo-->q31.3-->qter)[6]/46,XX [49].     

Image cytometry-based detection of aneuploidy by fluorescence in situ hybridization in suspension

Cytogenetic abnormalities are important diagnostic and prognostic criteria for hematologic malignancies. Karyotyping and fluorescence in situ hybridization (FISH) are the conventional methods by which these abnormalities are detected. The sensitivity of these microscopy-based methods is limited by the abundance of the abnormal cells in the samples and therefore these analyses are commonly not applicable to minimal residual disease (MRD) stages. A flow cytometry-based imaging approach was developed to detect chromosomal abnormalities following FISH in suspension (FISH-IS), which enables the automated analysis of several log-magnitude higher number of cells compared with the microscopy-based approaches. This study demonstrates the applicability of FISH-IS for detecting numerical chromosome aberrations, establishes accuracy, and sensitivity of detection compared with conventional FISH, and feasibility to study procured clinical samples of acute myeloid leukemia (AML). Male and female healthy donor peripheral blood mononuclear cells hybridized with combinations of chromosome enumeration probes (CEP) 8, X, and Y served as models for disomy, monosomy, and trisomy. The sensitivity of detection of monosomies and trisomies amongst 20,000 analyzed cells was determined to be 1% with a high level of precision. A high correlation (R(2) = 0.99) with conventional FISH analysis was found based on the parallel analysis of diagnostic samples procured from 10 AML patients with trisomy 8 (+8). Additionally, FISH-IS analysis of samples procured at the time of clinical remission demonstrated the presence of residual +8 cells indicating that this approach may be used to detect MRD and associated chromosomal defects. ? 2012 International Society for Advancement of Cytometry.     

A trisomic germ cell line and precocious chromatid segregation leads to recurrent trisomy 21 conception

A chromosomally normal 37-year-old woman was referred for preimplantation genetic diagnosis after having several conceptuses with trisomy 21. Segregation of chromosome 21 was assessed in unfertilised meiosis II oocytes and preimplantation embryos from PGD cycles using fluorescent in situ hybridisation (FISH). Of 7 preimplantation embryos, 5 were chromosomally abnormal with 4 having trisomy 21 and one being tetraploid. Of 4 oocytes, 3 had an abnormal chromosomal constitution with either an extra chromosome 21 or an extra chromatid 21. In one oocyte an extra chromatid 21 was detected in both the metaphase II complement and the first polar body providing the first direct evidence of a maternal trisomic germ cell line. Moreover, this result shows that the extra chromosome 21 can precociously divide into its two chromatids at the first meiotic division. Received: 9 September 1998 / Accepted: 26 November 1998     

胎儿稽留流产与染色体异常的关系及其影响因素分析

目的：探讨B超联合FISH实验室诊断技术分析胎儿稽留流产与染色体非整倍体关系并对其他影响因素进行综合分析。方法：采用FISH技术对广西267例B超诊断为稽留流产孕妇的胎儿绒毛组织行13,16,18,21,22,X,Y染色体数目检测,荧光显微镜下观察结果;采用SPSS13．0对相关数据进行统计分析。结果：267例稽留流产胎儿绒毛组织中,染色体数目异常95例,异常率35．6％,数目异常以三体最常见,其次为四体,少见部分单体;异常病例样本中存在多种染色体混合嵌合体现象,如混合嵌合三体（2n＋1／2n）,混合嵌合四体（2n＋2／2n）,混合嵌合单倍体、三体、四体（2n-1／2n＋l／2n＋2／2n）等;稽留流产与患者年龄、流产史、孕周具有显著相关性。结论：染色体数目异常与染色体混合嵌合均是稽留流产的重要原因,同时稽留流产发生与患者高龄、多次流产史、早期妊娠密切关系。     

Arginase deficiency manifesting delayed clinical sequelae and induction of a kidney arginase isozyme

De novo chromosome structural abnormalities cannot always be diagnosed by the use of standard cytogenetic techniques. We applied a previously developed chromosome-band-specific painting method to the diagnosis of such rearrangements. The diagnostic procedures consisted of microdissection of an aberrant chromosomal region of a given patient, polymerase chain reaction (PCR) amplification of the dissected chromosomal DNA, and subsequent competitive fluorescence in situ hybridization (FISH) using the PCR products as a probe pool on metaphase chromosomes from the patient and/or a karyotypically normal person. With this strategy, we studied 6 de novo rearrangements (6p+, 6q+, 9p+, 17p+, +mar, and +mar) in 6 patients. These rearrangements had been seen by conventional banding but their origin could not be identified. In all 6 patients, we successfully ascertained the origin. Using an aberrant region-specific probe pool, FISH signals appeared on both the aberrant region and a region of another specific chromosome pair. A reverse probe pool that was generated through the microdissection of normal chromosomes at a candidate region for the origin of the aberration hybridized with both the aberrant and the candidate regions. We thus diagnosed one patient with 17p+ as having trisomy for 14q32-qter, one with 9p+ as having trisomy for 12pter-p12, one with 6q+ as having a tandem duplication (trisomy) of a 6q23-q25 segment, one with 6p+ as having a tandem duplication (trisomy) of a 6p23-q21.3 segment, one with a supernumerary metacentric marker chromosome as having tetrasomy for 18pter-cen, and the last with an additional small marker chromosome as having trisomy for 18p11.1 (or p11.2)-q11.2. The present targeted chromosome-band-painting method provides the simple and rapid preparation of a probe pool for region-specific FISH, and is useful for the diagnosis of chromosome abnormalities of unknown origin.     

Adjusting interphase FISH results in epithelial tissue sections to whole cell complement

OBJECTIVE: To derive an equation to compensate for the discrepancies between whole cell preparations and tissue sections for more accurate enumeration of fluorescence in situ hybridization (FISH) signals per cell. STUDY DESIGN: Mean centromere signal counts in touch preparations and corresponding 4-6-micron sections of paraffin-embedded tissue were calculated. Mean widest nuclear diameters were also determined from the tissue sections. The observed data were analyzed to define the volumetric relationships between tissue sections and whole cell preparations. RESULTS: Analysis of results from six lung specimens yielded an equation that approximates whole versus sectioned nuclear volume and permits accurate quantification of mean FISH signal count in histologic sections, as follows: [formula: see text].     

Characterisation of a 19-year-old "long-term survivor" with Edwards syndrome

Trisomy 18 is the second most frequent autosomal aneuploidy affecting about 1 in 8,000 new-borns. Similar to trisomy 13 more than 90% of the patients die within the first year. Main causes of death are failure of vital organ function, in most cases of brain, heart, kidney, and gut, sometimes combined with severe infections. The degree to which essential organs are affected at birth and the clinical course differ considerably. Unknown genetic factors and various environmental effects are most likely involved. A less severe course of Edwards syndrome can be caused by a partial trisomy due to a deletion of the extra chromosome 18 or somatic mosaicism with a trisomic and a normal cell-line in the patient. In this report conventional chromosome analysis, FISH, and QF-PCR have been performed on a 19-year-old female patient with trisomy 18 to investigate a large number of cells including non-mitotic cells from various different tissues. This study supports evidence for an apparently pure form of trisomy 18 in this "long-living" patient with Edwards syndrome.     

Prenatal diagnosis of tetrasomy 18p using multiplex fluorescent PCR and comparison with a variety of techniques

We report genetic characterization of isochromosome 18p using a combination of cytogenetic and molecular genetic methods, including multiplex fluorescent PCR. The patient was referred for chorionic villus sampling (CVS) due to advanced maternal age and maternal anxiety. The placental karyotype was 47,XX,+mar, with the marker having the appearance of a small supernumerary isochromosome. Because differentiating between isochromosomes and other structural rearrangements is normally very difficult, a variety of genetic tests including fluorescence in situ hybridization (FISH), PCR, and multiplex fluorescent PCR were undertaken to determine chromosomal origin and copy number and, thus, allow accurate diagnosis of the corresponding syndrome. FISH determined that the marker chromosome contained chromosome 18 material. PCR of a variety of short tandem repeats (STRs) confirmed that there was at least one extra copy of the maternal 18p material. However, neither FISH nor PCR could accurately determine copy number. Multiplex fluorescent PCR (MF-PCR) of STRs simultaneously determined that: (1) the marker included 18p material; (2) the marker was maternal in origin; (3) allele copy number indicated tetrasomy; and (4) contamination of the sample could be ruled out. Results were also rapid with accurate diagnosis of the syndrome tetrasomy 18p possible within 5 hours.     

人乳头状瘤病毒诱导人胚食管上皮永生化细胞恶性转化

为了证实ＨＰＶ１８Ｅ６Ｅ７基因诱导的人胚食管上皮永生化细胞（ＳＨＥＥ）６１代（ＳＨＥＥ６１）部份细胞（ＳＨＥＥ６１Ａ）已经恶性转化,以寻找监控细胞早期恶性转化的方法。对培养的ＳＨＥＥ第１０代（ＳＨＥＥ１０）和６１代（ＳＨＥＥ６１）细胞,用光学是为微镜和电子显微镜观察细胞形态及生长形式;用流工细胞仪分析细胞周期;用染色体Ｇ带核型分析和间期核染色体１、７、８号着丝粒探针荧光原位杂交９ＦＩＳＨ）检测细胞     

Acrocallosal syndrome in a child with de novo inverted tandem duplication of 12p11.2-p13.3.

Report on the child of normal unrelated parents presenting the typical features of acrocallosal syndrome (craniofacial dysmorphy, mental deficiency, convulsive disorder, agenesis of corpus callosum, preaxial polydactyly "hallux duplex" of both feet, and in addition diabetes insipidus) in which a mirror duplication of nearly the entire short arm of chromosome 12 was discovered. Since the symptomatology of trisomy and tetrasomy 12p shows some overlap with acrocallosal syndrome a common origin of the monogenic disorder and the chromosomal phenotypes is discussed.     

Trisomy of medial 15q as result of an analphoid supernumerary ring chromosome detected by CGH and FISH

We report a 21-year-old patient with a de novo mosaic, analphoid ring of chromosome 15q22.2-->q24.1. The clinical features of this patient are mild and include tall stature, obesity, striae distensae in the hypogastrium, malocclusion and bilateral gynecomastia with scarce glandular tissue. M-FISH and FISH using a chromosome 15 painting probe indicated that the ring is of chromosome 15 origin. Further CGH analysis and FISH with the PML locus-specific probe demonstrated that the extra material derived from the medial part of the long arm of chromosome 15, including two bands, q22 and q23. Additionally, FISH with BAC probes specific for 15q allowed for a localization of the breakpoints at 15q22.2 and 15q24.1, distal to clones RP11-30M4 and RP11-500O23 respectively. We discuss the relationship between the patient's genotype and phenotype comparing it to reported cases of trisomy of medial 15q.     

Molecular studies of chromosomal mosaicism: relative frequency of chromosome gain or loss and possible role of cell selection.

Studies of uniparental disomy and origin of nonmosaic trisomies indicate that both gain and loss of a chromosome can occur after fertilization. It is therefore of interest to determine both the relative frequency with which gain or loss can contribute to chromosomal mosaicism and whether these frequencies are influenced by selective factors. Thirty-two mosaic cases were examined with molecular markers, to try to determine which was the primary and which was the secondary cell line: 16 cases of disomy/trisomy mosaicism (5 trisomy 8, 2 trisomy 13, 1 trisomy 18, 4 trisomy 21, and 4 involving the X chromosome), 14 cases of 45,X/46,XX, and 2 cases of 45,X/47,XXX. Of the 14 cases of mosaic 45,X/46,XX, chromosome loss from a normal disomic fertilization predominated, supporting the hypothesis that 45,X might be compatible with survival only when the 45,X cell line arises relatively late in development. Most cases of disomy/trisomy mosaicism involving chromosomes 13, 18, 21, and X were also frequently associated with somatic loss of one (or more) chromosome, in these cases from a trisomic fertilization. By contrast, four of the five trisomy 8 cases were consistent with a somatic gain of a chromosome 8 during development from a normal zygote. It is possible that survival of trisomy 8 is also much more likely when the aneuploid cell line arises relatively late in development.     

Congenital anomalies and developmental delay in a boy with double chromosome 6 derived supernumerary marker

The frequency of small supernumerary marker chromosomes has been estimated to approximately 0.45 per 1000 newborns. They are usually seen as single marker chromosomes in a mosaic state. Two cytogenetically identical markers have been observed only occasionally. We report on a boy, with congenital heart defect, neonatal hypotonia, hypogenitalism, delayed psychomotor development and mild dysmorphic facial features. The GTG karyotype performed on peripheral blood lymphocytes revealed a mosaic male karyotype with three cell lines. One cell line had a normal karyotype. In the other two either single or double chromosome 6 derived supernumerary markers were present, leading to partial trisomy or partial tetrasomy of chromosome 6, respectively.     

Uniparentale Disomien und Mosaike

Of the various mechanisms of formation of uniparental disomy (UPD) discussed in the literature, the mechanism of trisomy rescue is mostly prone to mosaicism from a trisomy cell line and from a disomy 46, XN uniparental cell line. Therefore, low level or undetected mosaicism has been assumed for a significant number of UPD cases. The clinical consequences of trisomy/UPD mosaicism probably depend on the chromosome involved and the proportional content in individual tissues. As the trisomy cell line of some mosaics might have a disadvantage in biological selection it might not be detected in routine lymphocyte investigations. For evaluation of the clinical relevance in the case of an imprinted chromosome the associated imprinting disorder must also be considered. In a postnatal setting analysis of UPD is indicated in the case of clinical, cytogenetic and molecular data. In the prenatal setting genetic counseling of the parents should be offered prior to any laboratory testing. In total, the impact of mosaicism associated with UPD has to consider the affected chromosome, the associated phenotype, the mechanism of formation and the laboratory method used.     

Proliferation enhancement by spontaneous multiplication of chromosome 7 in rheumatic synovial cells in vitro

Mosaic trisomy of chromosome 7 is known to occur in a variety of non-neoplastic hyperproliferative disorders. In long-term cell cultures established from rheumatic synovium with mosaic trisomy 7, we observed a continuous increase in the proportion of cells with trisomy 7 to over 50% by the 10th in vitro passage. Simultaneous in situ hybridization with a repetitive chromosome-7-specific DNA probe and fluorescent Ki-67 labelling showed a strong correlation between trisomy 7 and an elevated proliferation index in cultured rheumatic synovial cells. Moreover, we observed a fraction of rapidly proliferating cells with up to eight copies of chromosome 7 as the sole cytogenetic change. Frequent somatic pairing of centromeres of two chromosomes 7 in interphase nuclei suggests either atypical non-disjunction with a persisting centromere or selective endoreduplication of chromosome 7.