Pure proximal deletion of chromosome 21 and kyphosis.

We report on two unrelated patients with a proximal deletion of the long arm of chromosome 21. The deletion encompassed 14.5Mb of DNA. Molecular studies showed that the two telomeric breakpoints were within the same DNA clone (BAC RP11-56D12). The centromeric breakpoints, however, were separated by only 250kb of DNA (BAC RP11-645E14 and RP11-324B9). The phenotype observed in the two patients was very different, as patient 2, who had the largest deletion, had severe kyphosis not observed in patient 1. Previous studies have identified a 6Mb region of chromosome 21 associated with severe kyphosis. Interestingly, this region overlaps the 250kb segment deleted in patient 2. We suggest that one gene (NT011512.4) located in this small overlapping region might be responsible for severe kyphosis.

Establishment and characterization of a human parathyroid carcinoma derived cell line.

Parathyroid carcinoma is a rare neoplasia associated with PTH-dependent hypercalcaemia. It is infrequent in primary hyperparathyroidism (HPT) and very rarely associated with uremic HPT. A continuous cell line named Pt.Kich-1 from a parathyroid carcinoma diagnosed in a patient with secondary hyperparathyroidism (II° HPTH) was established and maintained in vitro for more than 60 passages. The cells were characterized for their immunophenotypic and endocrine characteristics as well as for their functional status after treatment by the extracellular [Ca(2+)]e, and the calcium homeostasis regulator 1α,25 (OH)2D3. The cytogenetic features were established by the R-banding. The Pt.Kich-1 cultures show an aspect of admixed epithelial/mesenchymal like cells with a doubling time between 96 and 112h. The cells are immunoreactive for cytokeratin (60%), EMA (26%), vimentin (46%), E-cadherin (32%), and synaptophysin (16%), while chromogranin A was not detected. Hypotetraploid karyotype containing large chromosomal markers and double minute chromosomes was identified in 30% of the metaphases. Treatment of Pt.Kich-1 cells with 1.0mM, 1.5mM, and 1.7mM of extracellular [Ca(2+)]e increased the DNA synthesis, while the calcium homeostasis regulator, the 1α,25 (OH)2D3, at 10(-9)-10(-7)M inhibited the cell growth. The levels of PTH measured in the medium of early cultures ranging between 547 and 610pg/µg of DNA declined during the passages to a level between 6 and 12pg/µg of DNA. No effect on the PTH release by the Pt.Kich-1 cells was observed after treatment with the all-trans (ATRA) and 9-cis retinoic acid differentiation inducers. The described in vitro cellular model can serve as a useful tool to study the pathogenesis of parathyroid carcinoma and to improve the knowledge of the molecular mechanisms involved in the control of gland sensitivity to [Ca(2+)]e leading to PTH synthesis and secretion.

A peptidase gene in chromosome 8q is disrupted by a balanced translocation in a duane syndrome patient.

PURPOSE: To identify the gene disrupted by a de novo reciprocal balanced translocation t(6;8)(q26;q13) in a patient with Duane retraction syndrome (DURS). The break point in chromosome arm 8q is positioned within the DURS1 critical region. METHODS: Fluorescence in situ hybridization (FISH) analysis using cosmid and BAC clones covering the DURS1 locus was performed to define the break point position and its relationship with expressed sequence tags (ESTs) in the region. Once the interrupted gene was identified, the full-length cDNA was sequenced and the genomic organization defined. Eighteen patients with sporadic DURS without cytogenetic abnormalities involving the DURS1 region were screened for point mutations in the candidate DURS1 gene. RESULTS: A carboxypeptidase gene (CPAH) was directly interrupted between the first and second exons in a patient with DURS who carried a de novo reciprocal balanced translocation t(6;8)(q26;q13) involving the DURS1 region on chromosome arm 8q13. The gene was transcribed in at least two alternative mRNA forms, with different start and stop codons. CONCLUSIONS: The CPAH gene was interrupted in a patient with DURS carrying a translocation break point in the DURS1 region on chromosome 8q13. CPAH is therefore a likely candidate for this abnormality, even if the possibility that other genes are involved, either by direct effects on transcription units present in the first CPAH intron or by position effects, cannot be ruled out. Functional studies of the influence of this gene on the morphogenesis of eye muscles and their innervation may clarify this question.

Molecular cytogenetic aberrations in CD30+ anaplastic large cell lymphoma cell lines.

Anaplastic large cell lymphomas (ALCL) in children represent a heterogeneous group of neoplasms with regard to the cell lineages involved. The chromosomal 5q35 breakpoint (bp) and the expression of the NPM/ALK fusion gene are the most remarkable molecular cytogenetic features of these malignancies. To identify new locations of ALCL-related oncogenes, comparative genomic hybridization (CGH) was applied to three ALCL cell lines (SU-DHL-1, Karpas 299, and DEL) exhibiting the 5q35 bp and expressing the NPM/ALK transcript. The CGH profiles were compared with those obtained with DNA from U937, HL-60 cells, and altered lymph nodes from two children with ALCL. Significant DNA copy number gains and/or losses were observed on several chromosomes in all ALCL cell lines. Distinct amplicons were detected on 1q21 approximately q44 (DEL), 7q12 (SU-DHL-1), and 1q12 approximately q22 (Karpas 299) regions. The NPM/ALK fusion gene was confirmed by fluorescence in situ hybridization (FISH) analysis in more than 80% of interphase nuclei and metaphase spreads. Enhanced expression of TGF-beta2 and c-MET candidate genes located at the amplified regions was revealed in DEL and SU-DHL1 cell lines by Northern blot analysis. These findings delineate chromosomal imbalances in ALCL-derived cell lines in parallel with high level of amplification covering target DNA sequences, which could play a role in ALCL pathogenesis.

Molecular cytogenetic anomalies and phenotype alterations in a newly established cell line from Wilms tumor with diffuse anaplasia.

The novel continuous cell line WT-Pe.1 was established in vitro from Wilms tumor with histological features of diffuse anaplasia. The cultures grew as poorly differentiated epithelial-like cells with pleomorphic polygonal shapes and formation of typical monolayers. WT-Pe.1 cells were immunoreactive for cytokeratin, vimentin, laminin, villin, CD10, and CD24 proteins. Conventional cytogenetic analysis by RHG-banding revealed a hypotriploid karyotype with numerous abnormalities including ring chromosomes, double-minutes, homogeneous staining regions, radial structures, dicentrics, and several marker chromosomes. Comparative genomic hybridization analysis revealed DNA copy numbers losses on chromosome segments 1p, 3p, 6q, 9q34.1 approximately q34.3, 11q24 approximately q25, 14q12 approximately qter, 16q, 18q, and 22q11 approximately q13; gain of genomic material was localized on chromosome arms 1q, 4p, 6q, and 7p and the entire chromosome 12. With DNA from the original tumor, copy number losses were detected on chromosomes 1p, 14q, 16q, 17q, and 22q and gains were observed on 1q, 4p, 8q, 12p, 12q, and chromosome 14p. Copy number amplifications of distinct loci were found on 1q21.1 and 4p15.3, as well as an elevated copy number of cyclin D2 (CCND2) and cyclin D associated kinase (CDK4) genes on chromosome 12 (confirmed by fluorescence in situ hybridization).

Early pattern of epilepsy in the ring chromosome 20 syndrome.

PURPOSE: The characteristics of epilepsy in ring chromosome 20 have been reported in adolescents and adults. The mode of onset most often remains imprecise. To clarify this onset period, we studied the early-onset features in our personal series and in the reported pediatric cases. METHODS: Our series comprises one child with an onset of epilepsy in the neonatal period and five others with an onset before age 8 years. The cases in the literature with an epilepsy onset before 8 years also were reviewed. RESULTS: Seizures in the neonatal period were described as motor seizures. Our personal patient with a neonatal onset had severe psychomotor delay. In both infancy and early childhood, the EEG showed no interictal frontal localization of the anomalies, and no long-lasting seizure was recorded. Seizures with terror and hallucinations usually appeared from about age 4 years. It is not before the age of 8 years that the usual interictal EEG pattern appeared of rhythmic theta slow-waves activity with spikes predominating in frontal areas described in adolescence and adulthood. The interictal EEG showed 1- to 2-Hz delta slow waves and spike-and-waves predominating in frontal areas, but no physiologic activity. CONCLUSIONS: In ring 20 chromosome, specific epilepsy features are lacking in the neonate, but the whole phenotype shows a more severe expression in terms of mental delay. The characteristic frontal EEG pattern and ictal terror do not appear before age 4 to 5 years.

DOR-1, A novel CD10+ stromal cell line derived from progressive Langerhans cell histiocytosis of bone.

BACKGROUND: Langerhans cell histiocytosis (LCH) is granulomatous proliferative disorder characterized by the presence of activated Langerhans cells admixed with macrophages, lymphocytes, and eosinophils. In an effort to obtain an LCH ex vivo model, we succeeded in establishing the DOR-1 cell line from an LCH lesion of bone in a 3-year-old girl. PROCEDURE: The DOR-1 cell line was established from a CD1a immunoreactive LCH lesion of bone maintained in long-term cell culture. The phenotypic characteristics were assessed by immuno-cytochemistry and fluorescence activated cell sorter (FACS) analysis. Cytogenetic analysis was performed by RHG-banding that was supplemented by fluorescence in situ hybridization (FISH). RESULTS: The DOR-1 cells grew in vitro as a poorly differentiated mesenchymal-like cells with a doubling time between 72 and 96 hr. The cells exhibited pleomorphism and consistent immuno-reactivity for CD10 (50%), CD13 (55%), CD68 (65%), and CD117 (70%) while CD1a, Langerin and HLA-DR were not detected. By RHG-banding, several aberrant chromosomes were detected including the t (9; 17) (p23; p13) translocation and a pair of long dicentric marker chromosomes indicating clonal abnormality. Functionally, exposure to 33 nM 12-O-tetradecanoyl phorbol mirystate-13-acetate (TPA) induced DOR-1 cell differentiation with appearance of cytoplasmic extensions. CONCLUSIONS: The DOR-1 cell line exhibits distinct immuno-cytochemical features and carries the t (9; 17) (p23; p13) translocation suggesting involvement of stromal-like cell lineage in LCH initiation and progression.

Molecular cytogenetic aberrations in autosomal dominant polycystic kidney disease tissue.

Autosomal dominant polycystic kidney disease (ADPKD) is a genetically heterogeneous disorder characterized by focal cyst formation from any part of the nephron. The molecular bases include germinal mutation of either PKD1 or PKD2 genes, enhanced expression of several protooncogenes, alteration of the TGF-alpha/EGF/EGF receptor (EGFR) axis, and disturbed regulation of proliferative/apoptosis pathways. To identify new locations of ADPKD related oncogenes and/or tumor suppressor genes (TSG), comparative genomic hybridization (CGH) and loss of heterozygosity (LOH) analyses were performed for a series of individual cysts (n = 24) from eight polycystic kidneys. By CGH, imbalances were detected predominantly on chromosomes 1p, 9q, 16p, 19, and 22q in all tissues. DNA copy number gain was seen on chromosomes 3q and 4q in five samples. The CGH data were supplemented by LOH analysis using 83 polymorphic microsatellite markers distributed along chromosomes 1, 9, 16, 19, and 22. The highest frequency of LOH was found on the 1p35-36 and 16p13.3 segments in cysts from seven samples. Allelic losses on 9q were detected in six, whereas deletions at 19p13 and 22q11 bands were observed in three polycystic kidneys. These results indicate that the deleted chromosomal regions may contain genes important in ADPKD initiation and progression.