Lukrecija Brecevic

Intrachromosomal triplication of 15q11-q13.

A 7 year old girl with intrachromosomal triplication 46,XX,-15,+der(15)(pter-->q13::q13-->q11::q11-->qter) resulting in tetrasomy of 15q11-q13 is reported. Fluorescence in situ hybridisation confirmed that the tetrasomic region included the entire segment normally deleted in Prader-Willi and Angelman syndrome patients, and breakpoints were similar to those reported in two tandem duplications of 15q11-q13. The middle repeat was inverted, suggesting a possible origin through an inverted duplication intermediate. Microsatellite analysis showed that the rearrangement was of maternal origin and involved both maternal homologues. Clinical findings included multiple minor anomalies (a fistula over the glabella, epicanthic folds, downward slanting palpebral fissures, ptosis of the upper lids, strabismus, a broad and bulbous tip of the nose, and small hands and feet), motor and mental retardation, a seizure disorder, and limited verbal abilities. In addition, immunological examination disclosed a selective immunodeficiency. The overall phenotype did not clearly resemble that of cases with tetrasomy 15pter-q13 associated with an extra inv dup(15)(pter-->q13:q13-->pter) chromosome. The latter aberration causes more severe mental deficit and intractable seizures, but less marked phenotypic alterations, although some overlap in mild facial dysmorphic features is present. A number of features common to Angelman syndrome were also observed in the patient.

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.

Phenotype of the Williams-Beuren syndrome associated with hemizygosity at the elastin locus

To correlate presence or absence of a 7q11 microdeletion with the clinical picture of the Williams-Beuren syndrome (WBS), we investigated 29 patients with a clinical diagnosis of WBS or WBS-like features, aged 1–30 years, using molecular analysis and/or fluorescent in situ hybridization (FISH). Deletions at 7q11 were found in 75% of the patients (22 out of 29). Nine deletions occurred on a paternal, and ten on a maternal chromosome; three deletions were demonstrated by FISH only, and parental origin could thus not be determined. All deletion patients aged between 2 years and puberty displayed a distinct pattern of facial features (including periorbital fullness, short nose with flat bridge, wide mouth, and full lips and cheeks), the characteristic outgoing social behaviour, as well as moderate growth and mental retardation. Twothirds (15 out of 22) had a cardiovascular malformation, but only one third (7 of 22) had supravalvular aortic stenosis (SVAS). A stellate iris pattern was also present in one-third of the patients only. In the four adult patients with 7q11 deletions, there was prominence of the lower lip whereas fullness of cheeks and periorbital tissue was not seen.ConclusionThis study confirms that WBS has a unique clinical picture which can be diagnosed clinically, but also shows that the relative frequency of individual features may have been overemphasized in the past, and that a minority of patients may exist who are clinically indistinguishable from WBS but who appear to have no deletion at 7q11.

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.

Somatic Mosaicism in Cases with Small Supernumerary Marker Chromosomes

Somatic mosaicism is something that is observed in everyday lives of cytogeneticists. Chromosome instability is one of the leading causes of large-scale genome variation analyzable since the correct human chromosome number was established in 1956. Somatic mosaicism is also a well-known fact to be present in cases with small supernumerary marker chromosomes (sSMC), i.e. karyotypes of 47,+mar/46. In this study, the data available in the literature were collected concerning the frequency mosaicism in different subgroups of patients with sSMC. Of 3124 cases with sSMC 1626 (52%) present with somatic mosaicism. Some groups like patients with Emanuel-, cat-eye- or i(18p)- syndrome only tend rarely to develop mosaicism, while in Pallister-Killian syndrome every patient is mosaic. In general, acrocentric and non-acrocentric derived sSMCs are differently susceptible to mosaicism; non-acrocentric derived ones are hereby the less stable ones. Even though, in the overwhelming majority of the cases, somatic mosaicism does not have any detectable clinical effects, there are rare cases with altered clinical outcomes due to mosaicism. This is extremely important for prenatal genetic counseling. Overall, as mosaicism is something to be considered in at least every second sSMC case, array-CGH studies cannot be offered as a screening test to reliably detect this kind of chromosomal aberration, as low level mosaic cases and cryptic mosaics are missed by that.

Multiple congenital anomalies including the Rieger eye malformation in a boy with interstitial deletion of (4) (q25-->q27) secondary to a balanced insertion in his normal father: evidence for haplotype insufficiency causing the Rieger malformation.

A 7 year old boy with minor facial anomalies, the Rieger eye malformation, reduced vision, genital anomalies, and severe mental retardation had deletion of the segment 4q24-->q26. His phenotypically normal father had a balanced insertion of that segment into the distal long arm of chromosome 6: 46,XY,ins(6;4)(q26;q24q26). Microsatellite loci flanking the RIEG gene on 4q25 were deleted giving indirect evidence of deletion of this locus. This finding and the normal ocular findings in the insertion carrier father show that haplotype insufficiency can cause the Rieger eye malformation.

Concomitant Amplif ication 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.

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.

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.

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.