Identification of twelve new RFLP-markers on chromosome 22q11-qter

Summary We have previously identified and regionally localized 195 chromosome-22-specific DNA markers. We now report restriction fragment length polymorphisms detected by 9 phage markers mapped to 22q11-q12, two cosmid clones mapped to 22q12-q13 and one plasmid mapped to 22q13-qter. These markers may be useful tools for mapping disease genes such as the NF2 locus, on chromosome 22.     

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.     

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.     

Maternal origin of extra marker chromosome 1Q31.1-qter and 13pter-q12.12 in a child with dysmorhic features

Maternal origin of extra marker chromosome Iq31.l-qter and 13pter-q12.12 in a child with dysmorphic features: We describe a twenty-days-old female child with dysmorphic features and chromosomal analysis with GTG-banding revealed an extra marker chromosome. Fluorescence in situ hybridization (FISH) study of extra marker chromosome confirmed to be maternal der(13) chromosome, contained 1q31.1-qter and 13pter-q11 chromosomal material and resulted from a maternal balanced translocation t(1;13)(q31.1; q12.12). The child had the majority of trisomy 1q clinical features: dysmorphic features, micropthalmia, high arched palate, long philthrum, micrognathia, hypertelorism, low set ears, short sternum, overlapping fingers, valgus of wrists, right knee and ankle joints were in flexion contractures. This is the first case of trisomy 1q with an extra marker chromosome which also contained chromosome 13pter-q12.12 material.     

Large duplication 4q25-q34 with mild clinical effect

We report on a 5-year-old Tunisian boy with particular dysmorphic features and mild mental retardation limited in delayed and poor language acquisition. Cytogenetic analysis using RHG banding and FISH using whole chromosome four painting probe showed a partial duplication in the long arm of chromosome four. Locus specific probes and CGH confirmed the presence of a 'pure' partial trisomy 4q due to de novo direct tandem dup(4)(q25q34). Comparative analysis of our case with those published previously, suggests that region 4q31-q33 may be involved in the development of the 4q characteristic dysmorphic features and the distal band 4q35 may be involved in the development of microcephaly and severe mental and growth retardation.     

Chromosomal localization of 9 KOX zinc finger genes: physical linkages suggest clustering of KOX genes on chromosomes 12, 16, and 19

Nine KOX zinc finger genes were localized on four human chromosomes by in situ hybridization of cDNA probes to metaphase chromosomes. KOX1 (ZNF10), KOX11 (ZNF18), and KOX12 (ZNF19) were mapped to chromosome bands 12q24.33, 17p13-p12, and 16q22-q23, respectively. Six other KOX genes were localized on chromosome 19: KOX6 (ZNF14) and KOX13 (ZNF20) to 19p13.3-p13.2, KOX5 (ZNF13) and KOX22 (ZNF27) to 19q13.2-qter, and KOX24 (ZNF28) and KOX28 (ZNF30) to 19q13.4. Pulsed field gel electrophoresis experiments showed that the pairs of KOX genes found on the chromosome bands 12q24.33, 16q22-q23, 19p13.3-p13.2, or 19q13.3-qter lie within 200–300 kb DNA fragments. This suggests the existence of KOX gene clusters on these chromosomal bands.     

Chromosomal imbalances in primary and metastatic melanomas revealed by comparative genomic hybridization

Characteristic genetic changes underlying the metastatic progression of malignant melanoma is incompletely understood. The goal of our study was to explore specific chromosomal alterations associated with the aggressive behavior of this neoplasm. Comparative genomic hybridization was performed to screen and compare genomic imbalances present in primary and metastatic melanomas. Sixteen primary and 12 metastatic specimens were analyzed. We found that the pattern of chromosomal aberrations is similar in the two subgroups; however, alterations present only in primary and/or metastatic tumors were also discovered. The mean number of genetic changes was 6.3 (range 1-14) in primary and 7.8 (range 1-16) in metastatic lesions. Frequent losses involved 9p and 10q, whereas gains most often occurred at 1q, 6p, 7q, and 8q. Distinct, high-level amplifications were mapped to 1p12-p21 and 1p22-p31 in both tumor types. Amplification of 4q12-q13.1, 7q21.3-qter and 8q23-qter were detected only in primary tumors. The 20q13-qter amplicon was present in a metastatic tumor. The number of genetic alterations were significantly higher in primary tumors which developed metastases within one year after the surgery compared to tumors without metastasis during this time period. Fluorescence in situ hybridization with centromeric and locus-specific probes was applied to validate CGH results on a subset of tumors. Comparison of FISH and CGH data gave good correlation. The aggressive behavior of melanoma is associated with accumulation of multiple genetic alterations. Chromosome regions, which differ in the primary and metastatic lesions, may represent potential targets to identify metastases-related chromosomal alterations.     

De novo balanced complex chromosome rearrangement (CCR) involving chromosome 8, 11 and 16 in a boy with mild developmental delay and psychotic disorder

Congenital Complex Chromosome rearrangements (CCRs) compatible with life are rare in humans. We report a de novo CCR involving chromosomes 8, 11 and 16 with 4 breakpoints in a patient with mild dysmorphic features, acquisition delay and psychotic disorder. Conventional cytogenetic analysis revealed an apparently balanced 8;16 translocation. Further FISH analysis with WCP 8 and WCP 16 probes revealed the presence of a third chromosome involved in the translocation. The multicolour karyotype confirmed the complexity of the rearrangement and showed that the derivative chromosome 8 was composed of 3 distinct segments derived from chromosomes 8, 16 and 11. The breakpoints of this complex rearrangement were located at 8q21, 11q14, 11q23 and 16q12. Comparative genomic hybridization (CGH) and array-CGH were performed to investigate the possibility of any genomic imbalance as a result of the complex rearrangement. No imbalance was detected by these two techniques. Our study showed: i) the necessity to confirm reciprocal translocations with FISH using painting probes, particularly when the karyotype resolution is weak; ii) the usefulness of multicolour karyotype for the characterization of structural chromosomal rearrangements, particularly when they are complex; iii) the usefulness of CGH and array-CGH in cases of abnormal phenotype and apparently balanced rearrangement in order to explore the breakpoints and to detect additional imbalances.     

A de novo unbalanced reciprocal translocation identified as paternal in origin in the Prader-Willi syndrome

Summary Interstitial cytogenetic deletions involving the paternally derived chromosome 15q11–13 have been described in patients with the Prader-Willi syndrome (PWS). We report a child with PWS and a de novo unbalanced karyotype –45,XY,–9,–15,+der(9)t(9;15)(q34;q13). Molecular studies with the DNA probe pML34 confirmed that only a single Prader Willi critical region (PWCR: 15q11.2-q12) copy was present. Hybridisation of patient and parental DNA with the multi-allelic probe CMW1, which maps to pter-15q13, showed that the chromosome involved in the translocation was paternal in origin. This is the first example of a paternally-derived PWCR allele loss caused by an unbalanced translocation that has arisen de novo.     

Molecular mapping of the Edwards syndrome phenotype to two noncontiguous regions on chromosome 18.

In an effort to identify regions on chromosome 18 that may be critical in the appearance of the Edwards syndrome phenotype, we have analyzed six patients with partial duplication of chromosome 18. Four of the patients have duplications involving the distal half of 18q (18q21.1-qter) and are very mildly affected. The remaining two patients have most of 18q (18q12.1-qter) duplicated, are severely affected, and have been diagnosed with Edwards syndrome. We have employed FISH, using DNA probes from a chromosome 18-specific library, for the precise determination of the duplicated material in each of these patients. The clinical features and the extent of the chromosomal duplication in these patients were compared with four previously reported partial trisomy 18 patients, to identify regions of chromosome 18 that may be responsible for certain clinical features of trisomy 18. The comparative analysis confirmed that there is no single region on 18q that is sufficient to produce the trisomy 18 phenotype and identified two regions on 18q that may work in conjunction to produce the Edwards syndrome phenotype. In addition, correlative analysis indicates that duplication of 18q12.3-q22.1 may be associated with more severe mental retardation in trisomy 18 individuals.     

Characterization of double minute chromosomes’ DNA content in a human high grade astrocytoma cell line by using comparative genomic hybridization and fluorescence in situ hybridization

The presence of double minute chromosomes (dmin) in cancer cells is known to be correlated with gene amplifications. In human high grade astrocytomas or glioblastomas, about 50% of cytogenetically characterized cases display dmin. G5 is a cell line which has been established from a human glioblastoma containing multiple dmin. In order to identify the DNA content of these dmin, three techniques were successively used: conventional cytogenetic analysis, comparative genomic hybridization (CGH), and fluorescent in situ hybridization (FISH). The karyotype of G5 cells showed numerical chromosome changes (hypertriploidy), several marker chromosomes, and multiple dmin. CGH experiments detected two strong DNA amplification areas located in 9p21-22 and 9p24, as well as an underrepresentation of chromosomes 6, 10, 11, 13, 14, and 18q. By using FISH with a chromosome 9-specific painting probe to metaphase chromosomes of the G5 cell line, dmin were shown to contain DNA sequences originating from chromosome 9. This study demonstrates the usefulness of a combination of classical karyotyping, CGH, and FISH to identify the chromosomal origin of amplified DNA sequences in dmin. Received: 30 October 1994 / Revised: 25 February 1996     

Detection of an unbalanced t(4;15) by FISH in a child with multiple congenital anomalies

Detection of an unbalanced t(4;15) by FISH in a child with multiple congenital anomalies: In this report, we present the clinical history and findings in a 6-month-old male with multiple congenital anomalies, developmental delay, and an initial male karyotype with 4q+. The origin of the additional segment on 4q was unequivocally established by fluorescence in situ hybridization (FISH). Whole chromosome probe for chromosome 4 and chromosome 15-specific a-satellite probe were used. The karyotype was demonstrated to be 46,XY,der(4), t(4;15)(q35;?),inv(9)(p13q13). To the best of our knowledge the above cytogenetic abnormalities with these clinical findings have not been described previously. This case further demonstrates the advantage of FISH in the identification of anomalous chromosome regions and breakpoints.     

14个染色体区带特异性探针池的构建

本文运用人类染色体显微切割和ＰＣＲ技术,成功地构建了１４个染色体区带特异性探针池,并通过染色体原位杂交证明它们均分别来源于相应的被切割的染色体区带。     

Genomic imbalances associated with methotrexate resistance in human osteosarcoma cell lines detected by comparative genomic hybridization-based techniques

Methotrexate (MTX) is one of the most important drugs for osteosarcoma (OS) treatment. To identify genetic aberrations associated with the development of MTX resistance in OS cells, in addition to the previously reported expression changes of dihydrofolate reductase (DHFR) and reduced folate carrier (RFC) genes, comparative genomic hybridization (CGH)-based techniques were used. The direct comparison between MTX-resistant variants of U-2OS or Saos-2 human OS cell lines with their respective parental cell lines by CGH on chromosomes revealed that development of MTX resistance was associated with gain of the chromosomal regions 5q12-q15 and 11q14-qter in U-2OS variants, and with gain of 8q22-qter in Saos-2 variants. Further analyses by CGH on microarrays demonstrated a progressively increasing gain of mixed lineage leukemia (MLL) gene (11q23) in U-2OS MTX-resistant variants, which was also confirmed by fluorescence in situ hybridization (FISH), in addition to gain of FGR (1p36), amplification/overexpression of DHFR, and slight decrease of RFC expression. In Saos-2 MTX-resistant variants, gain of MYC (8q24.12-q24.13) was detected, together with a remarkable decrease of RFC expression. Further analyses of DHFR, MLL, MYC, and RFC gene status in four additional human OS cell lines revealed that only gain of DHFR and MLL were associated with an inherent lower sensitivity to MTX. These data demonstrate that genetic analyses with complementary techniques are helpful for the identification of new candidate genes, which might be considered for an early identification of MTX unresponsive tumors.     

Characterization of supernumerary rings and giant marker chromosomes in well-differentiated lipomatous tumors by a combination of G-banding,CGH, M-FISH,and chromosome- and locus-specific FISH

Supernumerary ring chromosomes and/or giant marker chromosomes are often seen in soft-tissue tumors of low-grade or borderline malignancy, such as well-differentiated liposarcomas or atypical lipomas. Classic cytogenetic banding techniques have proved insufficient to identify the genomic composition and structure of such rings and markers, but fluorescent in situ hybridization (FISH) studies have shown that they consist mainly of amplified material from chromosome 12, more specifically from bands 12q13-->q15. We have used the new FISH-based screening techniques comparative genomic hybridization (CGH) and multicolor-FISH (M-FISH) in combination with G-banding and analysis by chromosome- and locus-specific fluorescent in situ probes to examine in detail the karyotypic characteristics of 22 lipomatous tumors, most of them classified histologically as well-differentiated liposarcomas, selected because they had been shown to harbor rings and/or marker chromosomes. M-FISH, in contrast to G- banding, was found to be informative with regard to the chromosomal origin of the rings and other markers present, whereas CGH and hybridizations with locus-specific probes helped identify which subchromosomal regions were involved. We found that chromosome bands 12q15-->q21 were always gained, with 12q15-->q21 being amplified (i.e., a green-to-red ratio >2 by CGH) in 14 of 22 tumors. In three tumors, two distinct but close amplicons in 12q could be identified, corresponding to bands 12q13-->q15 and 12q21. The genomic segment 1q21-->q23 was gained in 12 cases, reaching the level of amplification in seven. Bands 6q24 and 7p15, whose pathogenetic involvement in liposarcomas has not been reported previously, were gained in three cases each. In addition, the rings and giant markers often contained interspersed sequences from several other chromosomes that did not give an equally clear impression of being nonrandomly involved.     

Regional Assignment of Conserved Reference Loci Anchors Unassigned Linkage and Syntenic Groups to Ovine Chromosomes

Seven loci that have been previously mapped to human and mouse chromosomes have now been regionally assigned to six sheep chromosomes. Nerve growth factor β (NGFB), antigen CD3 ζ polypeptide (CD3Z), inhibin β A (INHBA), estrogen receptor (ESR), rhodopsin (RHO), insulin-like growth factor 2 (IGF2), and myelin basic protein (MBP) were mapped by in situ hybridization to sheep chromosomes 1p24-p21, 1p14-p11, 4q26-q31, 8q25-q27, 19q23-qter, 21q21-qter, and 23q11-q12.3, respectively. ESR, RHO, IGF2, and MBP are the first markers to be assigned to their respective sheep chromosomes. These new data allow the previously unassigned sheep linkage groups H, J, K, and S to be provisionally assigned to chromosomes 21, 19, 4, and 8, respectively. The unassigned sheep syntenic groups U8 and U13 are provisionally assigned to sheep chromosomes 8 and 21, respectively. The new assignments support the emerging picture that there is extensive conservation of human chromosomal segments in the sheep and cattle genomes. The position of another evolutionary breakpoint on human chromosome 1q is suggested.     

A dysmorphic newborn with 45,X,der(1)inv(1)(p13;qter)t(Y;1)(pter-->q11;p13),-Y de novo karyotype

A dysmorphic newborn with 45,x,der(1)inv(1)(p13;qter)t(y;1)(pter-->q11;p13),-Y de novo karyotype: Y/autosome translocations are very rare chromosomal rearrangements. In most cases, the long arm of the Y chromosome is translocated onto an autosome and most patients are referred because of male infertility. Y/1 translocations are very rare, and have been reported in seven patients so far. Pericentric inversions may be seen in all chromosomes and are not associated with phenotypic abnormalities. Here we report a 6-day old male baby with prenatal growth retardation, frontal bossing, hypertelorism, micrognathia, cleft soft palate, absent uvula, hypospadias, simian line in both hands and hammer toes. Cytogenetic analysis was performed with GTG-banding, C-banding and FISH analysis containing X centromeric probe, Yq12-qter locus specific probe and whole chromosome Y probe. An unbalanced Y/1 translocation was diagnosed: 45,X,der(1)inv(1)(p13;qter)t(Y;1)(pter-->q11;p13),-Y.     

Constitutional duplication 11q23 de novo involving the MLL gene

We report a child with mental retardation, brain anomalies and congenital heart defect. His karyotype, after G-banding and FISH with a whole chromosome probe for chromosome 11 and a locus-specific probe for the MLL gene, was 46,XY,dup(11)(q23q23).ish dup(11)(q23q23)(wcp11+, MLL++) de novo; i.e., he had a pure partial 11q23 duplication. Clinical and cytogenetic findings of the present case were compared with the 7 previously reported cases with pure partial trisomy 11q; in 6/8 cases the region 11q23 was involved. We conclude that the scarce number of cases and their heterogeneity do not allow to establish a reliable genotype-phenotype correlation.     

Ring chromosome 15: characterization by array CGH

Ring chromosome 15 [r(15)] is an uncommon finding with less than 50 patients reported. Precise genotype–phenotype correlations are problematic because of the difficulties in determining the extent of euchromatic loss, the level of mosaicism, and the influence of the timing of ascertainment. We report two discordant examples of r(15) patients. In the first case, prenatal diagnosis of a de novo r(15) was made during the second trimester: mos 46,XX,r(15)(p11.2q26)[32]/45,XX,-15[13]/47,XX,r(15)(p11.2q26)x2[3]/46,XX,dic r(15)(p11.2q26p11.2q26[1]/46,XX[2]. Postnatal follow-up revealed extremely small stature, heart defects, and developmental delay. Patient 2 was a 31-year-old short-statured female who was living independently: 46,XX,r(15)(p11q26). Both cases showed loss of the 15q subtelomeric region by fluorescence in situ hybridization (FISH). To investigate the discordance in phenotypes between the two patients, we undertook array comparative genomic hybridization (array CGH) analyses to more fully characterize the deletions associated with these otherwise structurally indistinguishable r(15) chromosomes from conventional cytogenetic analyses and fluorescence in situ hybridization (FISH) studies. By array CGH, patient 1 showed deletion of multiple contiguous clones predicting an approximately 6 Mb deletion of distal 15q. In contrast, patient 2 showed loss of just the 15q subtelomeric clone and an interstitial clone by array CGH confirming that the severity of the phenotype correlated with the size of the deletion at the molecular level. These cases illustrate the utility of array CGH characterization for determining the size of the associated deletion in ring chromosomes and for facilitating phenotype–genotype correlations.