Anneke Y. van der Veen

FISH and array-CGH analysis of a complex chromosome 3 aberration suggests that loss of CNTN4 and CRBN contributes to mental retardation in 3pter deletions.

Imbalances of 3p telomeric sequences cause 3p− and trisomy 3p syndrome, respectively, showing distinct, but also shared clinical features. No causative genes have been identified in trisomy 3p patients, but for the 3p− syndrome, there is growing evidence that monosomy for one or more of four genes at 3pter, CHL1, CNTN4, CRBN, and MEGAP/srGAP3, may play a causative role. We describe here an analysis of a complex chromosome 3p aberration in a severely mentally retarded patient that revealed two adjacent segments with different copy number gains and a distal deletion. The deletion in this patient included the loci for CHL1, CNTN4, and CRBN, and narrowed the critical segment associated with the 3p− syndrome to 1.5 Mb, including the loci for CNTN4 and CRBN. We speculate that the deletion contributes more to this patients phenotype than the gains that were observed. We suggest that 3p− syndrome associated features are primarily caused by loss of CNTN4 and CRBN, with loss of CHL1 probably having an additional detrimental effect on the cognitive functioning of the present patient.

Normal FHIT transcripts in renal cell cancer- and lung cancer-derived cell lines, including a cell line with a homozygous deletion in the FRA3B region

The recently identified FHIT gene encompasses the FRA3B region and the breakpoint of a constitutive t(3;8) occurring in a family with hereditary renal cell cancer. Occurrence of aberrant transcripts in different types of tumours has led to the suggestion that FHIT might play a critical role in the development of various types of cancer. We have analyzed the gene and its transcripts in lung cancers and renal cell cancer‐derived cell lines. A lung adenocarcinoma cell line, GLC‐A2, appeared to have a homozygous deletion in intron 5 of FHIT. RT‐PCR analysis revealed a normal‐sized PCR product in all of the cell lines, including GLC‐A2. A number of them had an additional aberrant product. Analysis of a great number of control cell lines and tissues showed that the majority of these also had aberrant PCR products in addition to a normal‐sized PCR product. Different specimens of the same cell type showed variable additional RT‐PCR products. Normal‐sized PCR products had a sequence identical to the FHIT sequence. PCR products longer than normal had insertions of different sizes at different positions. With three exceptions, PCR products shorter than normal represented FHIT sequences missing one or more entire exons. Thus, the presence of aberrant transcripts is not cancer‐specific. Conceivably, sequences responsible for the instability of the FRA3B region are being transcribed into FHIT pre‐mRNA and may cause the abnormal splicing and processing of the transcripts. Genes Chromosom. Cancer 19:220–227, 1997.

Analysis of a new homozygous deletion in the tumor suppressor region at 3p12.3 reveals two novel intronic noncoding RNA genes

Homozygous deletions or loss of heterozygosity (LOH) at human chromosome band 3p12 are consistent features of lung and other malignancies, suggesting the presence of a tumor suppressor gene(s) (TSG) at this location. Only one gene has been cloned thus far from the overlapping region deleted in lung and breast cancer cell lines U2020, NCI H2198, and HCC38. It is DUTT1 (Deleted in U Twenty Twenty), also known as ROBO1, FLJ21882, and SAX3, according to HUGO. DUTT1, the human ortholog of the fly gene ROBO, has homology with NCAM proteins. Extensive analyses of DUTT1 in lung cancer have not revealed any mutations, suggesting that another gene(s) at this location could be of importance in lung cancer initiation and progression. Here, we report the discovery of a new, small, homozygous deletion in the small cell lung cancer (SCLC) cell line GLC20, nested in the overlapping, critical region. The deletion was delineated using several polymorphic markers and three overlapping P1 phage clones. Fiber‐FISH experiments revealed the deletion was approximately 130 kb. Comparative genomic sequence analysis uncovered short sequence elements highly conserved among mammalian genomes and the chicken genome. The discovery of two EST clusters within the deleted region led to the isolation of two noncoding RNA (ncRNA) genes. These were subsequently found differentially expressed in various tumors when compared to their normal tissues. The ncRNA and other highly conserved sequence elements in the deleted region may represent miRNA targets of importance in cancer initiation or progression. Published 2006 Wiley‐Liss, Inc.

Infantile and adult testicular germ cell tumors: a different pathogenesis?

Most adult testicular germ cell tumors have a characteristic chromosomal abnormality that is an isochromosome 12p [i(12p)]. Furthermore, these tumors are characterized by a chromosome number in the triploid range and gains and losses of (parts of) specific chromosomes. Cytogenetic investigation of three cases of infantile testicular germ cell tumors, all diagnosed as yolk sac tumors, revealed highly abnormal karyotypes. We found one case to be diploid; the other two cases were in the hypertriploid/hypotetraploid range. Structural abnormalities of chromosomes 1, 3, and 6 were recurrent and no i(12p) was found. Our results, together with data from the literature, suggest that infantile and adult testicular germ cell tumors have a different origin and pathogenetic pathway. Aberrations of chromosomes 1, 3, and 6 may play an important role in the pathogenesis of infantile testicular yolk sac tumors.

Cytogenetics of a malignant ovarian germ-cell tumor

Cytogenetic investigation of a malignant ovarian tumor diagnosed as a mixed germ‐cell tumor, composed of extensive choriocarcinoma and foci of yolk‐sac tumor, revealed a highly abnormal chromosomal pattern. We found a chromosome number in the hypertriploid/hypotetraploid range, and several clonal structural abnormalities, including 2 copies of an isochromosome 12p. We showed that the chromosomal pattern of this ovarian tumor is very similar to that of testicular germ‐cell tumors. This finding, together with reported cytogenetic data of malignant ovarian germ‐cell tumors, supports the hypothesis that ovarian and testicular germ‐cell tumors are strongly related entities that may have a similar origin and pathogenetic pathway. Int. J. Cancer 77:217–218, 1998.© 1998 Wiley‐Liss, Inc.

LOCALIZATION OF AMPLIFIED C-MYC AND N-MYC IN SMALL CELL LUNG-CANCER CELL-LINES

In this study 12 small cell lung cancer cell lines were tested for amplification of myc oncogenes, the location of amplified sequences, and the possible correlation between number of dmin and degree of amplification in dmin-containing lines. C-myc appeared to be amplified in four cell lines, and N-myc amplification was detected in two cell lines. No amplification of L-myc was found. The degree of amplification in the different cell lines varied between 20X and 100X. The cell lines with myc amplification appeared to contain numerous dmin, although in one cell line they occurred in only 10% of the cells. The other cells in this line contained a homogeneously staining region (HSR). In situ hybridization was carried out to find the location of the amplification. In four cell llines the amplified myc genes were found to be located on the dmin. In the cell line with the HSR in most cells and dmin in a minority of its cells, amplification was found both at the HSR and on the dmin. In one cell line the myc sequences seemed to be dispersed through the genome. The ratio between the average number of dmin per cell and the degree of amplification did not vary considerably between the cell lines, with one exception. In that cell line the number of dmin exceeded the number of myc sequences by about one order of magnitude. Apparently, the population of dmin in this cell was heterogeneous and amplified myc genes were only present on a subpopulation.

Loss of the Y-Chromosome in the Primary Metastasis of a Male Sex Cord Stromal Tumor: Pathogenetic Implications

The first published chromosomal pattern of the retroperitoneal lymph node metastasis of a malignant gonadal stroma cell tumor of the adult testis is presented. Karyotyping showed structural chromosomal abnormalities and loss of the Y-chromosome. This loss was confirmed in primary tumor and metastasis using fluorescence in situ hybridization (FISH). The characteristic chromosomal abnormality of adult testicular germ cell tumors, an i(12p), was not present. The results are compared with other data of testicular and ovarian sex cord stromal tumors. From the comparison of the male tumors, it is concluded that loss of the Y-chromosome might have a pathogenetic significance.

Allelic gains and losses in distinct regions of chromosome 6 in gastric carcinoma

In gastric cancer, alterations in the long arm of chromosome 6 are a frequent event. Two regions of heterozygous loss have been described: 6q16.3-6q23 and 6q26-6q27. We have evaluated by microsatellite and FISH analyses the 6q status of three cell lines that we established from primary gastric carcinomas xenografted in nude mice, in order to elucidate the underlying mechanisms of 6q alterations. Alterations of the long arm of chromosome 6 were found in all three xenografts and corresponding cell lines. Allelic imbalance (AI) was found in the three cases, by microsatellite analysis. Fluorescence in situ hybridization analyses clearly demonstrated a duplication of the larger part of the 6q arm in all three cell lines. Two of the cell lines and the corresponding xenografts showed, in addition, complete loss of one of the parental alleles at the terminal part of 6q. Thus, the AI observed along the long arm of chromosome 6 is represented by gain of alleles in one distinct chromosomal segment and loss of alleles in another distinct segment.

Functional analysis of lung tumor suppressor activity at 3p21.3.

The early and frequent occurrence of deletions at 3p21.3 in lung cancer has led to the consideration of this chromosomal region as a lung cancer (LUCA) critical region with tumor suppressor activity. We covered this 19 genes‐containing region with overlapping P1 artificial chromosomes (PACs), in which genes are likely accompanied by their own promoters or other regulatory sequences. With these PACs we transfected cells from a small cell lung cancer (SCLC) cell line which readily caused tumors in nude mice. Per PAC we selected two cell clones with a low number of PAC copies integrated at a single genomic site. The selected clones were s.c. injected into nude mice to investigate whether the integrated genes suppressed the tumor‐inducing capacity of the original SCLC cell line. We could demonstrate PAC‐specific gene expression in the transfected cells. All of the PAC integration sites were different. It appeared that introduction of a PAC or even an empty PAC vector causes some chromosomal instability, which in principle may either promote or inhibit cell growth. However, both cell clones with integration of the same PAC from the centromeric part of the LUCA region in different genomic sites were the sole pair of clones that caused smaller tumors than did the original SCLC cell line. This suggests that rather than the induced chromosomal instability, the DNA sequence of that PAC, which in addition to two protein‐encoding genes contains at least one potential miRNA gene, is responsible for the tumor suppressor activity.

Localization of DNA probes with tight linkage to the cystic fibrosis locus by in situ hybridization using fibroblasts with a 7q22 deletion

SummaryFive DNA probes known to originate from the region 7q22-q31 were sublocalized by in situ hybridization to metaphase preparations of fibroblasts having besides a normal chromosome 7, a homologue 7 with an apparent interstitial deletion of a large part of band q22. A flow cytometric chromosome analysis confirmed a loss of material from one of the homologues of chromosome 7. Four of the probes, B79a, 7C22,metH, and pJ3.11, have been shown to be closely linked to the cystic fibrosis (CF) locus. We localized probes B79a and 7C22 to the part of 7q22 involved in the deletion, whereasmetH and pJ3.11 could be assigned to band 7q31. Probe pJu28, for which polymorphisms have not yet been described, also appeared to derive from the latter band. Since pJ3.11 andmetH are most tightly linked to the CF locus, this disease locus is indirectly assigned to 7q31. A comparison of our findings with linkage data suggests a discrepancy between genetic and physical distances in the region 7q22-q31.