Human Ring Chromosomes - New Insights for their Clinical Significance.

Twenty-nine as yet unreported ring chromosomes were characterized in detail by cytogenetic and molecular techniques. For FISH (fluorescence in situ hybridization) previously published high resolution approaches such as multicolor banding (MCB), subcentromere-specific multi-color-FISH (cenM-FISH) and two to three-color-FISH applying locus-specific probes were used. Overall, ring chromosome derived from chromosomes 4 (one case), 10 (one case), 13 (five cases), 14, (three cases), 18 (two cases), 21 (eight cases), 22 (three cases), X (five cases) and Y (one case) were studied. Eight cases were detected prenatally, eight due developmental delay and dysmorphic signs, and nine in connection with infertility and/or Turner syndrome. In general, this report together with data from the literature, supports the idea that ring chromosome patients fall into two groups: group one with (severe) clinical signs and symptoms due to the ring chromosome and group two with no obvious clinical problems apart from infertility.

Multicolor FISH used for the characterization of small supernumerary marker chromosomes (sSMC) in commercially available immortalized cell lines.

There are only about 30 commercially available cell lines which include small supernumerary marker chromosomes (sSMC). As approximately 2.5 million people worldwide are carriers of an sSMC, this small number of immortalized cell lines is hard to understand. sSMC cell lines provide practically unlimited material for continuing studies e.g. to learn more about marker chromosome formation, or karyotypic evolution. To obtain information about their genetic content, in the present study we analyzed by FISH and multicolor-FISH approaches 19 sSMC cell lines obtained from the European Collection of Cell Cultures (ECACC). Microdissection and reverse painting, (sub-) centromere-specific multicolor-FISH (sub-)cenM-FISH, multicolor banding (MCB) and selected locus-specific FISH probes were applied. Thus, we were able to characterize comprehensively 14 out of 19 sSMC carrying cell lines; in the remaining five cases an sSMC could not be detected. Surprisingly, in six of the nine cell lines with sSMC previously characterized for their chromosomal origin by others, those results had to be revised. This has impact on the conclusions of previous studies, e.g. for uniparental disomy (UPD) in connection with sSMC.

Recombinant balanced and unbalanced translocations as a consequence of a balanced complex chromosomal rearrangement involving eight breakpoints in four chromosomes.

We report on a family with a balanced complex chromosomal rearrangement (CCR) involving eight breakpoints between chromosomes 6, 7, 18, and 21 in the father. All three sons inherited one derivative chromosome from the father and in addition each inherited a different recombinant chromosome resulting in a partial trisomy 6q in the first, an apparently balanced karyotype in the second, and a partial trisomy 7q in the third son. Fluorescence in situ hybridisation (FISH) and microsatellite analysis were essential for the identification of the breakpoints. In addition, the results were confirmed by a 24-colour FISH experiment using the spectral karyotyping (SKYtrade mark) system. Paternal origin of the de novo CCR in the father was demonstrated for the first time by haplotype analysis. This is the second report of a CCR leading to simpler but unbalanced translocations in offspring as a consequence of recombination during gametogenesis, and the first report of a family case of CCR exhibiting as many as eight breakpoints in the transmitting carrier. The initial prediction that viable offspring would be quite unlikely had to be revised after the birth of three children. Genetic counselling of carriers of balanced complex rearrangements has to consider a higher probability for unbalanced recombinations than has been so far commonly assumed.

Trisomy first, translocation second, uniparental disomy and partial trisomy third: a new mechanism for complex chromosomal aneuploidy.

A 2-year-old, short, microcephalic and developmentally retarded boy revealed a pattern of multiple minor anomalies, hypospadias and a dysplastic right kidney. Maternal age at delivery was 41 years. His karyotype showed two cell lines, one apparently normal, the other with a 1p+ chromosome. FISH examinations showed that the segment attached to 1p was from chromosome 16, and molecular investigations disclosed maternal heterodisomy 16, except for the segment (16)(pter-->p13.1) for which there was mosaicism between trisomy and uniparental disomy (UPD). Most likely, the zygote was trisomic for chromosome 16 due to a maternal meiosis I nondisjunction; a somatic rearrangement would have then occurred at an early postzygotic stage whereby a segment of the paternal chromosome 16 was translocated onto 1p. Subsequently, the paternal chromosomes 16 and 16p- had been lost in the normal and the translocation cell line, respectively. The chromosome aberration was detected secondary to the disclosure of maternal UPD 16 because of the demonstration of a paternal band at several loci on distal 16p. This case shows that chromosome aberrations may be formed in a more complicated manner than primarily assumed. Hence, the phenotype might also be due to underlying factors such as UPD or undetected mosaicism in addition to the more obvious implications of the chromosome rearrangement itself (e.g. partial trisomy).

Isochromosome 18p results from maternal meiosis II nondisjunction.

Microsatellite analysis with 13 microsatellites spread over 18p was performed to determine the origin of the marker chromosome in 9 patients with additional metacentric marker chromosomes. Phenotypes and banding patterns suggested that the markers were isochromosomes 18p. Maternal origin was determined in all 8 cases where both parents were available for study. Six cases showed 3 alleles (one paternal, one maternal each in single and double dose) of informative markers located close to the telomere while markers close to the centromere on 18p were reduced to homozygosity (one paternal allele in single dosage and one maternal allele presumably in triple dosage). A similar result was obtained in the patient with no parents available for examination. The other 2 patients were uninformative for maternal hetero- versus homozygosity, but at some loci the maternal band was clearly stronger than the paternal one whereas the opposite was never observed. Trisomy 18 differs from trisomy 21, XXX and XXY of maternal origin through a preponderance of meiosis II versus meiosis I nondisjunction. Thus, the results of our study and the advanced mean maternal age at delivery of patients with additional i(18p) indicate that in most if not all cases the marker chromosome originates from maternal meiosis II nondisjunction immediately followed by isochromosome formation in one of the 2 maternal chromosomes 18. Possible explanations of these results include a maternally imprinted gene on 18q with a lethal effect if the paternal homologue is lost and a mechanism through which nondisjunction in some cases could be connected with isochromosome formation.

Terminal deletion, del(1)(p36.3), detected through screening for terminal deletions in patients with unclassified malformation syndromes.

We report on a 4 year-old girl with a 1p36.3-pter deletion. Clinical findings included minor anomalies of face and distal limbs, patent ductus arteriosus, the Ebstein heart anomaly, and brain atrophy with seizures. Conventional GTG-banded chromosome analysis revealed a normal (46,XX) result. Subsequent analysis by fluorescent in situ hybridization (FISH) using distal probes demonstrated a deletion of 1p36.6-pter. Molecular investigations with microsatellite markers showed hemizygosity at three loci at 1p36.3 with loss of the paternal allele. The deletion of 1p36.3 is difficult to identify by banding alone; indeed, our patient represents the third reported case with a del(1)(p36.3) that was detected only after more detailed analysis. In all three cases the deletion was detected through screening of patients with multiple congenital anomalies/mental retardation syndromes suggestive of autosomal chromosome aberrations for subtelomeric submicroscopic deletions by means of FISH or microsatellite marker analysis. On the basis of these observations we highly recommend that FISH with a subtelomeric 1p probe be routinely performed in patients with similar facial phenotype, severe mental retardation and seizures, and a heart malformation, particularly the Ebstein anomaly.

Delimiting the use of comparative genomic hybridization in human myeloid neoplastic disorders.

Hematopoietic disorders can be used as a suitable tool of additional information on the actual resolving power of comparative genomic hybridization (CGH). Therefore, CGH examination was performed of DNA extracted from 23 acute and 15 chronic myeloproliferative disorders which had just been analyzed using classical cytogenetic techniques. In nearly all cases CGH analysis was repeated with reversely labeled probes. A Zeiss axioplan microscope was equipped with the ISIS 3 system for photometric evaluation of the CGH data. A main group was selected of 34 cases showing karyotypic mosaics when routinely diagnosed by classical cytogenetics. The grade of mosaicism was basically determined from the classical cytogenetic analysis and was additionally defined examining target anomalies by I-FISH analysis in 28 of the cases. The second group included 23 cases with deletions, and in 1 case another informative genomic imbalance could be analyzed. Every target anomaly irrespective of its type could be detected in all cases with an affected cell population equalling or exceeding about 25%, but in none was it below 23%. This value was the lowest and was found in a case, with CGH-detected 20q deletion. The smallest deletions of two bands on 20q which could visually be detected by CGH were estimated in the range of 5-7 Mb. CGH was also suitable to detect imbalances which were not clearly detected by routine cytogenetics. Reverse labelling, performed in nearly all cases, confirmed the result of the original CGH analysis. These data not only document the readiness and reliability of CGH studies on human leukemia, but also further contribute to a clearer definition of the limits of the resolving power of this technique.

Comparative genomic hybridization-aided unraveling of complex karyotypes in human hematopoietic neoplasias.

The information obtained by conventional cytogenetics (CC) in human leukemias is sometimes limited, in particular by complex karyotypes with many marker chromosomes. While CC is restricted to metaphases with a good quality, interphase fluorescence in situ hybridization (I-FISH) is also capable of analyzing specific anomalies in the interphase nuclei. Comparative genomic hybridization (CGH) gives additional information about the imbalanced karyotype changes in the whole genome. The aim of this study was to assess the contribution of CGH to the unraveling of complex GTG karyotypes, which are difficult to evaluate by banding analysis, and to compare these results with those by CC and FISH. Thirteen bone marrow samples and one sample obtained from peripheral blood of 13 leukemia patients were examined by CC, FISH and CGH. The GTG banding analysis showed complex karyotypes with many marker chromosomes. The most frequent abnormalities were numerical and structural aberrations on chromosomes 5 and 7. In 12 of the 14 samples, the CGH analysis was able to detect chromosomal imbalances with losses of material on chromosome 5 and 7 as the most frequent aberrations. In all 14 samples, additional FISH analyses were performed. For most of the studied neoplasias, a close correlation between CC, FISH and CGH data was observed. CGH was considerably helpful in adding additional information to classical karyotyping, if the low quality or number of metaphases was insufficient for a reliable CC analysis. Even in cases where whole chromosome painting could be applied, it added information on the breakpoints of the observed rearrangements. In only 2 of the studied 14 samples, neither CGH nor I-FISH could improve the result of karyotyping. CGH, nevertheless, can be regarded as a powerful additional technique in leukemias with unsuccessful CC, incomplete, or complex karyotypes with many marker chromosomes. A systematic analysis by three techniques such as CC, FISH and CGH guarantees an optimal genetic characterization of the neoplasias.

Concomitant amplification and expression of PAX7-FKHR and MYCN in a human rhabdomyosarcoma cell line carrying a cryptic t(1;13)(p36;q14).

Alveolar rhabdomyosarcoma (ARMS) is associated with the specific chromosomal translocation (2;13)(q35;q14) or its rarer variant t(1;13)(p36;q14), which produces the fusion gene PAX7-FKHR. Here we describe the human cell line RC2, derived from an ARMS, which harbors a cryptic t(1;13)(p36;q14) and concomitantly shows amplification of the PAX7-FKHR fusion gene and of the MYCN oncogene. The t(1;13) and MYCN oncogene were studied by standard cytogenetic analysis and molecular techniques. The reverse transcriptase polymerase chain reaction demonstrated the expression of PAX7-FKHR mRNA in RC2 cells, although karyotype analysis failed to demonstrate a t(1;13)(p36;q14) chromosomal translocation or a derivative 13 chromosome. Double minute chromosomes were detected in all the metaphases studied. Fluorescence in situ hybridization analysis revealed multiple copies of the PAX7-FKHR fusion gene localized exclusively on a subset of double minutes, whereas multiple copies of MYCN were identified on other double minute chromosomes. Southern-blot analysis demonstrated that RC2 cells contain approximately 20 copies of the MYCN oncogene. So far no continuous RMS cell line carrying the t(1;13)(p36;q14) has been described, and PAX7-FKHR and MYCN amplifications have always been reported to occur separately in rhabdomyosarcoma (RMS). The availability of an ARMS cell line that harbors the t(1;13)(p36;q14) constitutes a useful tool for further understanding the role of the PAX7-FKHR fusion gene in RMS oncogenesis and may improve knowledge of the possible relation between PAX7-FKHR and MYCN amplification.

The cytogenetic view of standard comparative genomic hybridization (CGH): deletions of 20q in human leukemia as a measure of the sensitivity of the technique.

BACKGROUND: The limits of the resolving power of comparative genomic hybridization (CGH) have been given as 10-20 Mbp if at least 50% of the studied neoplastic cell population carried the corresponding aberration. MATERIAL AND METHODS: Genomic DNA of five cases of hematologic neoplasias, in all of which--among other anomalies--deletions of different size of chromosome 20q were found by GTG banding and confirmed by FISH analyses, was subjected to CGH. RESULTS: CGH revealed four types of del(20q), and, in addition, detected a tiny terminal del(3p) in one of the cases. The size of the smallest deleted segment, clearly visible by eye on the CGH metaphase image, was estimated to range between 5 and 7 Mbp. CONCLUSION: Visual determination was shown to have a stronger resolving power in CGH than software used for the analysis in one case, while in another one, the results obtained from the ratio profiles would have been considered insignificant without the knowledge of the hybridization pattern on the corresponding CGH metaphase images. The potential of the standard CGH technique not only to detect, but visualize small segmental aneusomies as well, suggests that its resolution actually mirrors the resolution of banding techniques.

Mosaic supernumerary ring chromosome 1 in a three-generational family: 10-year follow-up report.

Additional small ring chromosome 1 is described with increasing rate of mosaicism in three generations. Ten years after the first examination, the mosaic rates in the patients were strikingly similar. An increase in the expression of phenotypic anomalies was also observed in the successive generations. FISH examinations following microdissection revealed signals which were positive for 1p13 and 1q21 indicating that the ring contained euchromatic segments on both ends. Additionally, array-CGH whole-genome analysis showed a single copy gain corresponding to band 1p12 to band 1q21-1 of chromosome 1 in the patients. The presence of euchromatic material from chromosome 1 in the ring suggests that the relationship between the cytogenetic findings and the clinical manifestation is likely causative. These unique observations might be explained by mitotic loss of the ring at early embryogenesis, and would indicate different mitotic vulnerability of certain chromosome abnormalities at early postzygotic stages versus later during development.

Precipitation of calcium phosphates from electrolyte solutions. V. The influence of citrate ions.

The influence of citrate ions on the precipitation of crystalline apatitic precipitates with low Ca/P molar ratios [octacalcium phosphate (OCP) and calcium-deficient apatites (DA) (system A)] and of the intercrystalline mixtures of calcium hydrogen phosphate dihydrate (DCPD) and DA (system B) was investigated. Samples were prepared by direct mixing of calcium chloride solutions (A, 6.10(-3) mol dm-3; B, 1.10(-1) mol dm-3) and sodium phosphate solutions (A, 6.10(-3) mol dm-3; B, 2.10(-2) mol dm-3) containing citrate (0-2.10(-3) mol dm-3) and preadjusted to pH 7.4. In the presence of citrate ions: (a) crystal growth of OCP and DA was slowed down; (b) habit modification of DCPD crystals occurred; and (c) equilibration in intercrystalline mixtures of DCPD and DA's was slowed down. All phenomena were caused by surface adsorption of negatively charged ions, most probably CaC6H5O7-, which is the prevalent calcium citrate species under the given experimental conditions. Habit modification of DCPD was induced by preferential adsorption at the (001) crystal plane.

Mechanism of antifoaming action of simethicone.

The mechanism of the foam-inhibiting action of simethicone, mainly used against flatulence, has been studied. Its effect on foaming systems containing synthetic gastric juice and a surface-active substance (anionic surfactant, cationic surfactant, soap solution) was quantitated by measuring the surface tension, foam stability and initial foam density. The effect of simethicone on each of the examined systems was the result of the combination of two actions: the drainage of liquid from foam films and the rupture of relatively thick liquid films. The mechanism of these actions may be described as the liquid drainage followed by bridging of the liquid film by polydimethylsiloxane droplets, helped by hydrophobic silica particles also present in the antifoaming agent, leading to the rupture of the film surface and air escape.

Formation and transformation of struvite and newberyite in aqueous solutions under conditions similar to physiological.

The precipitation of magnesium phosphates in the system MgCl(2)-NH(4)H(2)PO(4)-NaOH-H(2)O was studied at a wide range of reactant concentrations at 37 degrees C and an initial pH(i) of 7.4. Precipitates were aged for 24 h and characterized by means of optical and scanning electron microscopy, x-ray diffractometry, FT-IR spectroscopy and thermogravimetry. Struvite was found to precipitate in nearly the whole concentration region investigated. Pure newberyite and a mixture of struvite and newberyite were obtained in a narrow range only with a pH(24 h)<6.4. The transformation of struvite into newberyite was studied by following changes in the liquid and solid phases. Thus, the changes of pH and the content of struvite in the precipitate as a function of time were recorded. The influence of the initial pH(i) and temperature on the transformation process is discussed. Analysis of the experimental data suggests a solution mediated transformation process as a possible mechanism of struvite transformation.

Interstitial deletion, del(4)(q12q21.1), owing to de novo unbalanced translocation in a 2 year old girl: further evidence that the piebald trait maps to proximal 4q12.

A very short, microcephalic, and mentally retarded 2 year old girl showed minor anomalies including prominent occiput, delayed closure of the anterior fontanelle, high frontal hairline, prominent ears, upward slanting palpebral fissures, a small nose with bulbous tip, delayed tooth eruption and bone maturation, and short and tapering fingers and toes. She did not have a white forelock. Cytogenetic investigation disclosed a de novo unbalanced translocation between chromosomes 4 and 18 with deletion of 4q12-->q21.1. Molecular investigation showed lack of a paternal allele for the microsatellite markers D4S392 and D4S398. This case shows indirect evidence that the piebald gene maps to proximal 4q12.

Duplication of segment 1p21 following paternal insertional translocation, ins(6;1)(q25;p13.3p22.1).

A moderately mentally retarded 3 year old boy showed minor anomalies including a prominent forehead and flat occiput, exophthalmos, large and prominent ears, high arched palate, umbilical hernia, sacral dimple, and irregular position of the toes. Cardiac sonography disclosed a chorda running through the left ventricle. Cytogenetic investigation of the family showed a balanced insertional translocation of segment 1p13-->p22 into distal 6q in the father which had led, through unbalanced segregation, to duplication of 1p13.3-->p22.1 in the proband. Familial duplication of such a small interstitial segment of 1p has not been reported previously, and the paucity of abnormal physical findings in the proband compared to previous patients with a similar aberration is remarkable.

Chromosomal localization, genomic structure and characterization of the human gene and a retropseudogene for 6-pyruvoyltetrahydropterin synthase.

Autosomal recessive mutations in the 6-pyruvoyltetrahydropterin synthase (PTPS) gene are the most common reason for hyperphenylalaninemia due to tetrahydrobiopterin deficiency. We used the previously isolated PTPS cDNA as a probe and identified the human gene, PTS, located on chromosome 11q22.3-q23.3, and a retropseudogene, PTS-P1, assigned to 9p12-p13 (symbols approved by the human genome nomenclature committee). PTS-P1 has 74% similarity to the 3' portion of PTPS cDNA. The PTS gene spans about 8 kb and consists of 6 exons, as revealed by DNA-sequence analysis. This gene structure differs from that published previously which was reported to contain only two exons [Ashida, A., Owada, M. & Hatakeyama, K. (1994) Genomics 24,408-410]. By means of intron-specific primers, we amplified exon 3 from genomic DNA of a PTPS-deficient patient and found a mutation in the 3' acceptor splice site, which is responsible for skipping of exon 3.