Closely linked loci on the long arm of chromosome 13 flank a specific 2;13 translocation breakpoint in childhood rhabdomyosarcoma

A specific chromosomal translocation, t(2;13)(q35;q14), is present in tumor cells from about one-half of children with alveolar rhabdomyosarcoma, who generally have widely disseminated disease at diagnosis. Using a series of six DNA probes from five loci previously assigned to bands 13q12----q14, we have localized the translocation breakpoint on chromosome 13 by in situ hybridization. Each probe was used to examine metaphase spreads from two or more rhabdomyosarcoma cell lines that have the t(2;13), as well as from control lymphoblastoid cell metaphases. All six probes bound to chromosome 13q12----q14 in the control cell line, but showed no appreciable hybridization to other sites. With rhabdomyosarcoma metaphases, cDNA clones of the retinoblastoma susceptibility gene (RB1) and the esterase D gene (ESD), as well as the arbitrary genomic fragment 7D2 (D13S10), showed specific hybridization to the normal chromosome 13 and the der(2) marker, but not to the der(13). By contrast, the genomic fragments HU10 (D13S6) and 7F12 (D13S1) hybridized specifically to the normal chromosome 13 and the der(13), but not to the der(2). Thus, the breakpoint of this translocation lies distal to D13S6 and D13S1 and proximal to ESD, RB1, and D13S10. Our data indicate that the locus affected by the translocation breakpoint on chromosome 13, which we have termed RMS, is physically distinct from the RB1 locus and is, in fact, proximal to ESD, which others have placed at least 10(6) bp proximal to RB1. The consistent presence of the der(2) marker chromosome, coupled with occasional loss of the der(13), suggests that the RMS gene, or at least a critical component, moves to chromosome 2 in tumors with this translocation.     

Detection of retinoblastoma gene copy number in metaphase chromosomes and interphase nuclei by fluorescence in situ hybridization.

Fluorescence in situ hybridization (FISH) was applied to detect the copy number of the retinoblastoma (RB1) tumor suppressor gene in metaphase chromosomes and interphase nuclei. We used 14 lambda phage clones spanning the whole RB1 gene region as a probe and obtained a specific hybridization signal in normal metaphase chromosomes at 13q14. Normal interphase nuclei showed two RB1 signals in about 90% of cases, whereas two cell lines with cytogenetically defined deletions involving the RB1 gene showed only one hybridization signal in about 80% of the nuclei. Analogous changes were detected in metaphase chromosomes. Multicolor FISH with subsets of the phage clones allowed visualization of subregions within the 200-kb gene in interphase nuclei. Analysis of clinical breast cancer samples showed that most of the cells contained two copies of the RB1 gene, even when restriction fragment length polymorphism analysis showed loss of heterozygosity (LOH) at the RB1 locus. This indicates that LOH at the RB1 locus in breast cancer cells probably involves mechanisms other than physical deletion.     

Constitutional and somatic RB1 mutation spectrum in nonfamilial unilateral and bilateral retinoblastoma in India

An epidemiologic survey has indicated a comparatively high prevalence of retinoblastoma (Rb) in Asian countries. Recently, the development of preventive strategies in nonfamilial Rb has become a major goal. The present studies were designed for identification and characterization of constitutional and somatic RB1 gene mutations by conventional cytogenetics, fluorescent in situ hybridization (FISH) and polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP)-DNA sequencing. Of 34 patients 32 were nonfamilial and 2 were familial Rb. Maternal inheritance of del (13q14) was common. FISH was sensitive in detecting monoallelic RB1 deletion/deletion mosaicism as a first genetic hit in 20% of cases. Somatic and germline RB1 point mutations affected exons 3, 17, 20, and 21 and these were identified as novel mutations. Involvement of exon 20 as a predisposing mutation in sporadic unilateral retinoblastoma (URB) probably suggests the susceptibility of exon 20 to unknown etiologic factors in our population. A de novo RB1 deletion along with transmitted RB1 point mutation from an asymptomatic parent was identified as a unique predisposing RB1 mutation chimerism in a URB case that later evolved to bilateral retinoblastoma (BRB). The predisposing mutations such as del (13q), RB1 mono-allelic deletion and RB1 point mutation in sporadic Rb were de novo as well as transmitted mutations from asymptomatic/symptomatic parents. The RB1 mutation incidence was comparatively higher (25%) in nonfamilial Rb with emphasis on high prevalence in sporadic URB (18% versus 0%-9% in the literature series). The present studies demonstrated the efficacy of a multitechnique approach to detect various types of constitutional RB1 mutations such as RB1 deletion, deletion mosaicism, point mutation, mutation chimerism in patients of symptomatic/asymptomatic parents.     

De novo complex autosomal translocation involving chromosomes 8, 13 and 15 in a girl with a sporadic retinoblastoma

Summary We report a case of a 5-month-old female with sporadic monolateral retinoblastoma (RB) with a constitutional de novo complex autosomal translocation involving chromosomes 8, 13 and 15 resulting in a deletion of chromosome 13q14 confirmed by esterase D assay. The translocation of the terminal portion of chromosome 8 has been observed by in situ hybridization with c-myc and thyroglobulin probes.     

RB1 deletion in gonadoblastoma in an XY female

Cytogenetic studies of normal and tumor cells in a patient with gonadal dysgenesis and bilateral gonadoblastoma were performed. The karyotype was 46,XY in peripheral blood lymphocytes and skin fibroblasts. The conserved region of the SRY gene was detected by polymerase chain reaction amplification. Sequencing of this region did not reveal any alterations. A 46,XY chromosome constitution was observed in the right gonadoblastoma, but a partial deletion of chromosome 13 was present in the left tumor. This deletion included band 13q14, where the retinoblastoma gene is mapped. The study of the polymorphism of the variable number of tandem repeats region in intron 17 of the RB1 locus disclosed loss of heterozygosity in both the left tumor, which showed the deletion of chromosome 13, and in the right tumor, where no chromosome alterations of chromosome 13 were detected. In situ hybridization covering 130 kb of RB1 showed that a partial deletion of one of the RB1 alleles had occurred in the right tumor. Since the deletions affected different alleles in each tumor, independent events must have been involved in the development of the tumors. These findings point toward a significant role of RB1 in the development of gonadoblastoma. Received: 17 October 1995 / Accepted: 14 July 1997     

Eight closely linked loci place the Wilson disease locus within 13q14-q21

Wilson disease (WD) is an autosomal recessive disorder resulting in an accumulation of copper in the liver, brain, and other organs. The WD locus (WND) has previously been linked to esterase D (ESD) and localized to 13q14-22. With the large Centre d'Etude Polymorphisme Humain cohort, a refined map of DNA markers from this region was constructed, with the following locus order: D13S1-D13S21-D13S22-D13S10-ESD-RB-WND-D 13S26-D13S12-D13S2. A significant excess of male recombination was observed between D13S21 and D13S22. Intervals distal to D13S22 showed an excess of female recombination. When these markers were tested on 19 WD families from a variety of ethnic backgrounds, the two closest loci were shown to be RB and D13S26. The retinoblastoma gene locus (RB) was shown to be proximal to WND at a distance of 4.4 centimorgans (cM), and D13S26 was placed distal to WND at a distance of 4.0 cM. ESD was assigned proximally at a distance of 9.4 cM. In all families studied WND was linked to one or more of the loci ESD, RB, or D13S26.     

Molecular evidence that the esterase-D gene lies proximal to the retinoblastoma susceptibility locus in chromosome region 13q14

Summary Somatic cell hybrids have been created between transformed mouse 3T3 cells and fibroblasts from a retino-blatoma patient with normal red-cell esterase-D (ESD) levels and a constitutional deletion of chromosome region 13q14-q31. In one subclone, which has retained the deletion chromosome but not the homologous normal copy, we have demonstrated the presence of the human ESD gene sequence. The breakpoint in this patient therefore must have occurred between the ESD gene and the retinoblastoma (Rb) predisposition locus. We have also been able to demonstrate that the ESD gene lies proximally to be the Rb gene in region 13q14. The recently isolated 4.7R cDNA gene sequence was absent from the deletion-containing hybrid, a finding consistent with the hypothesis that this sequence represents the Rb gene itself.     

Esterase D and retinoblastoma gene loci are tightly linked to Wilson's disease in Chinese pedigrees from Taiwan

Summary Wilson's disease (WD) is a rare autosomal recessive disorder and has been mapped to the long arm of chromosome 13 (q14.1). We have analyzed the segregation of esterase D (ESD) and retinoblastoma (RB) gene loci in ten families of Chinese WD subjects living in Taiwan. The polymorphic information content (PIC) for ESD and RB was 0.18 and 0.31, respectively. We confirmed a tight linkage between these loci and WD with a lod score of 3.33 by multipoint linkage analysis. The data from this limited number of pedigrees also suggested the following order: centromere-WD-RB-ESD or centromere-ESD-RB-WD. ESD in conjunction with RB polymorphism would be useful in prenatal and presymptomatic diagnosis, as well as in carrier detection in informative pedigrees.     

荧光原位杂交技术在慢性淋巴细胞白血病遗传学异常检测中的应用

目的:分析在荧光原位杂交技术慢性淋巴细胞白血病遗传学异常检测中的应用,并分析相关指标在评价患者预后中的应用。方法:对我院收治的45例初诊CLL患者采用荧光原位杂交技术进行特异性探针D13S25(13q14.3)、RB1(13q14)、p53(17p13)、ATM(11q22.3)、以及CSP12(12号染色体3体)染色体标本检测,分析CLL患者遗传学异常的发生率。采用实时定量PCR检测miR-15a和miR-16-1与CLL患者遗传学异常的相关性。结果:45例CLL初诊患者中,荧光原位检测发现CLL遗传学异常37例,CLL遗传学异常率82.22%。其中d(13q14.3)遗传异常13例,d(13q14)遗传异常7例,d(11q22-23)遗传异常6例,d(17p13)遗传异常5例,12号染色体三体异常6例,遗传学异常多呈异质性。实时定量PCR检测发现miR-15a和miR-16-1与d(13q14)遗传异常显著相关。结论:荧光原位杂交技术是一种检测CLL遗传学异常的快速、灵敏方法,可以提高CLL遗传异常检出率。miR-15a和miR-16-1可以预测d(13q14)遗传异常CLL患者预后。     

Molecular-genetic analysis of two cases with retinoblastoma: Benefits for disease management

Effective counselling and management of retinoblastoma families using genetic information is presently practised in many parts of the world. We studied histopathological, chromosomal and molecular-genetic data of two retinoblastoma patients from India. The two patients, one with bilateral and the other with unilateral retinoblastoma, underwent complete ophthalmic examination, cytogenetic study, retinoblastoma gene (RB1) mutational analysis andRB1 promoter region methylation screening. In the bilateral retinoblastoma patient deletion of chromosome region 13q14 in peripheral blood lymphocytes and a hemizygous novel 8-bp deletion in exon 4 ofRB1 in tumour sample were observed. In the unilaterally affected patient CGA to TGA transition protein truncation mutations were observed in exons 8 and 14 ofRB1.     

Exclusion of the retinoblastoma gene and chromosome 13q as the site of a primary lesion for human breast cancer.

Chromosome 13q has been suggested as the site of a gene predisposing to human breast cancer, because loss of heterozygosity of alleles on this chromosome has been observed in some ductal breast tumors and because two breast cancer lines are altered at the retinoblastoma gene (RB1) at 13q14. To test this possibility, linkage of breast cancer susceptibility to 14 loci on chromosome 13q loci was assessed in extended families in which breast cancer is apparently inherited as an autosomal dominant trait. RB1 was excluded as the site of a breast cancer gene by a lod score of Z = -7.60 at close linkage for 13 families. Multipoint analysis yielded negative lod scores throughout the region between 13q12 and 13q34; over most of this distance, Z less than -2.0. Therefore, chromosome 13q appears to be excluded as the site of primary lesion for breast cancer in these families. In addition, comparison of tumor versus normal tissues of nonfamilial breast cancer patients revealed an alteration at the 5' end of RB1 in a mucoid carcinoma but no alterations of RB1 in five informative ductal adenocarcinomas. Linkage data and comparisons of tumor and normal tissues suggest that changes in the RBI locus either are secondary alterations associated with progression of some tumors or occur by chance.     

A quantitative trait locus on 13q14.2 for trunk strength.

Previous findings show strong evidence for the role of retinoblastoma (Rb) in myoblast proliferation and differentiation. However, it is not known whether variation in the retinoblastoma gene (RB1 ) is responsible for normal variation in human muscle strength. Therefore, a linkage analysis for quantitative traits was performed on 329 young male siblings from 146 families with muscle strength, using a polymorphic marker in RB1 (D13S153 on 13q14.2). Trunk strength, a general strength indicator that requires activation of large muscle groups, was measured on a Cybex TEF isokinetic dynamometer. We found evidence for linkage between locus D13S153 at 13q14.2 and several measurements of trunk flexion with LOD scores between 1.62 and 2.78 (.002< p <.0002). No evidence for linkage was found with trunk extension. This first exploration of the relationship between RB1 and human muscle strength through linkage analysis warrants efforts for further fine mapping of this region.     

Detection of submicroscopic deletions and a DNA polymorphism at the retinoblastoma locus

Summary DNA samples from 60 unrelated patients with retinoblastoma were screened by Southern blot hybridization using two probes that are closely linked to the retinoblastoma locus within human chromosome band 13q14. Seven of 44 patients with bilateral or multifocal unilateral retinoblastoma and one patient with unifocal unilateral retinoblastoma were found to have a heterozygous deletion for the anonymous DNA sequence H3-8. Three of the eight deletions did not include the esterase D locus and were undetectable by conventional cytogenetic analysis. The findings are compatible with the deletions being the cause of retinoblastoma in these cases and provide a basis for DNA diagnosis in nearly 20% of patients with bilateral and multifocal unilateral retinoblastoma. The H3-8 probe also detects a restriction fragment length polymorphism that is a useful genetic marker in some families.     

Studies on the human retinoblastoma susceptibility gene

The retinoblastoma susceptibility (RB) gene is unique among other cloned cancer genes because its causal role in a human cancer, retinoblastoma, was established by classical genetic methods before its isolation. Earlier hypotheses and experimental data suggested that inactivation of a gene in chromosome band 13q14 resulted in retinoblastoma formation. A gene in this region was identified as the RB gene on the basis of mutations found specifically in retinoblastoma tumors; however, its proposed biological activity in suppressing neoplasia has yet to be demonstrated. The RB gene product was identified as a nuclear phosphoprotein of 110 kD associated with DNA binding activity, suggesting that the RB protein may regulate other genes. Probes for the RB gene and gene product will be useful for genetic diagnosis of retinoblastoma susceptibility in affected families; for direct detection of mutant RB alleles; and, potentially, for genetic diagnosis of susceptibility to osteosarcoma and other tumors tentatively linked to RB-gene dysfunction. Continued study of the RB gene should yield further insight into mechanisms of oncogenesis, development, and gene regulation.     

A Yeast Artificial Chromosome Contig That Spans the RB1-D13S31 Interval on Human Chromosome 13 and Encompasses the Frequently Deleted Region in B-cell Chronic Lymphocytic Leukemia

Abnormalities involving chromosome 13 have been reported as the only cytogenetic change in B-cell chronic lymphocytic leukemia (BCLL). Deletions are the most common cytogenetic abnormality and always involve 13q14, but when translocations are seen, the consistent breakpoint is always in 13q14. It is now established that deletions, distal to the RB1 gene in 13q14, are invariably associated with these translocations. We have recently described the smallest such deletion from a series of rearrangements from these tumors isolated in somatic cell hybrids, which spans approximately 1 Mb. In this report, we present the results of a series of a chromosome walking experiments using YACs and have been able to span this small deletion, which must contain the gene that is frequently deleted in BCLL. Four probes from 13q14 (RBI-mgg15-D13S25-D13S31) were used to isolate corresponding YACs for each of the markers. The chromosomal location of these YACs was verified using FISH, which also demonstrated their nonchimeric nature. Vectorette end rescue was then used to demonstrate the overlap of the YACs and to isolate new clones to complete the contig. The extremes of the contig were shown to cross the chromosome 13 translocation breakpoints isolated in somatic cell hybrids that carry the derivatives of chromosome 13 involved in the smallest BCLL deletion. This YAC contig covers the entire deletion and will prove a valuable resource to begin isolating genes from this region. In addition, we have isolated YACs corresponding to the RB1 locus, which extends the contig over a 3.8-cM distance on the chromosome.     

CD138免疫磁珠细胞分选的FISH技术在多发性骨髓瘤诊断中的应用

为了分析CD138免疫磁珠细胞分选的染色体荧光原位杂交(FISH)技术在提高多发性骨髓瘤(MM)细胞遗传学异常检测敏感性的作用。本研究选取我院收治的30例确诊MM的患者为研究对象,分离骨髓单个核细胞,应用探针组合,同时采用2种方法进行细胞遗传学检测:实验组采用CD138免疫磁珠分选浆细胞后行荧光原位杂交技术(MACS-FISH)检测;对照组直接荧光原位杂交技术(D-FISH)检测。结果:30例MM患者,实验组采用CD138 MACS-FISH检出率为83.3%,对照组D-FISH法细胞遗传异常检出率为46.7%,两组差异具有统计学意义(p<0.05)。研究结果表明:分析不同类型的细胞遗传异常,MACS-FISH法1q21检出率为46.7%,RB1检出率为50.0%,Ig H检出率为70.0%,P53检出率为20.0%;D-FISH法检出率分别为23.3%,30.0%、36.7%、10.0%。通过细胞核型分析,30例MM患者中,发现5例患者为异常核型,仅为16.7%,其中1例患者为单一结构异常,复杂异常核型患者为4例。我们的研究结论表明:进行CD138免疫磁珠分选浆细胞的FISH技术在多发性骨髓瘤诊断应用中可显著提高细胞遗传学异常检测敏感性,具有临床推广应用的价值。     

Parental age and seasonal variation in the births of children with sporadic retinoblastoma: a mutation-epidemiologic study

Summary Statistical analysis of parental age data from 225 sporadic cases of bilateral retinoblastoma, plus ten sporadic cases of chromosome deletion or translocation involving 13q14 that was identified as of paternal origin, revealed no evidence of paternal or maternal age effect. Parental exposure to ionizing radiation or chemical mutagens, the effect of which is accumulated with advancing age, does not seem to play a major role in the production of germinal mutations at the responsible (RB) locus. Furthermore, analysis of variation in the month of birth of 753 children with sporadic unilateral retinoblastoma did not show any significant deviation from the controls or a cyclic trend. The occurrence of nonheritable retinoblastoma is not likely to be associated with certain viruses such as human adenovirus 12 whose activity varies markedly with season. These results, together with the fairly uniform pattern in the incidence of this tumor among different populations, suggest that most, if not all, cases of sporadic retinoblastoma are caused by some intrinsic biological mechanisms, and not by environmental mutagens that may vary with respect to time and place.     

New gene assignments to rabbit chromosomes; implications for chromosome evolution.

The genes for SOD1 and SOD2 (superoxide dismutases 1 and 2), RB1 (retinoblastoma), TYMS (thymidylate synthase), and TK1 (thymidine kinase) were mapped by in situ hybridization using biotinylated probes to rabbit chromosomes 6, 12, 8, 9, and 19, respectively. This confirms their proposed homoeologies with human chromosomes 21, 6, 13, 18, and 17, respectively, and provides additional information on the modification of these chromosomes during evolution.     

High rate of detection of 13q14 deletion mosaicism among retinoblastoma patients (using more extensive methods)

Summary Using a cell population with a high proportion of early mitotic cells and by examining more cells derived from peripheral lymphocytes, we found three cases with a 13q14 deletion mosaicism among fifteen retinoblastoma patients; one with a de novo 13/18 balanced translocation, and another with a monosomy 13(q13»q21.2 or 21.3). The three patients with a 13q14 deletion mosaicism had sporadic retinoblastoma (two had bilateral and one unilateral retinoblastoma). The results indicate that 13q14 deletion mosaicism plays a major role in the etiology of this tumor.     

No evidence for sequences structurally related to the RB1 gene in the human genome

Summary The retinoblastoma (RB1) gene is a ubiquitously expressed gene encoding a cell-cycle control protein. Inactivation of this gene plays a crucial role in the development of retinoblastoma, osteosarcoma, and other tumors. In a search for structurally related gene sequences we identified a 5.5-kb BamHI fragment strongly cross-hybridizing with the 5 end of the RB1 cDNA. Molecular cloning, in situ hybridization, restriction mapping, and sequence analysis identified this DNA segment as the 28S rRNA gene. The absence of other cross-hybridizing sequences suggests that the RB1 gene is not part of a structurally related gene family.