RB1 Gene Locus Research Articles

Dysplastic lipoma: potential diagnostic pitfall of using MDM2 RNA in situ hybridization to distinguish between lipoma and atypical lipomatous tumor

The distinction between lipoma and atypical lipomatous tumor can be challenging in some cases. While detection of MDM2 gene amplification via fluorescence in situ hybridization (FISH) has been well established as a diagnostic tool to distinguish atypical lipomatous tumor and well-differentiated liposarcoma from benign mimics, MDM2 RNA in situ hybridization (RNA-ISH) has recently been proposed as an alternative diagnostic assay. During clinical workup for lipomatous tumors using MDM2 RNA-ISH, we noticed several dysplastic lipomas that were positive for MDM2 RNA-ISH but negative for MDM2 amplification by FISH. In this study, we examined a series of 11 dysplastic lipomas, all confirmed to be negative for MDM2 amplification by FISH. Positive MDM2 RNA-ISH was noted in 10 (91%) dysplastic lipomas. Single-nucleotide polymorphism array on one dysplastic lipoma identified the presence of homozygous deletion of 13q, including the RB1 gene locus with no evidence of MDM2 copy number gain. Our findings on the discordance between MDM2 FISH and MDM2 RNA-ISH highlightthe potential utility and pitfalls of using MDM2 RNA-ISH in the distinction of atypical lipomatous tumor and related liposarcomas from dysplastic lipoma.

Detection of RB1 Gene Copy Number Variations Using a Multiplex Ligation-Dependent Probe Amplification Method.

Multiplex ligation-dependent probe amplification (MLPA) is based on simultaneous multiplex PCR of specific probes that hybridize to multiple different target DNA regions. The method can identify copy number changes, gross gene rearrangements, methylation patterns or even point mutations. MLPA has been a reliable approach to identify copy number changes in the clinical and research settings and is widely used for the screening and investigation of copy number variations and genomic aberrations of interest in various diseases. In this chapter the analysis of the copy number changes in the RB1 gene locus by MLPA is described.

Influence of clone and deletion size on outcome in chronic lymphocytic leukemia patients with an isolated deletion 13q in a population-based analysis in British Columbia, Canada.

Deletion of the long arm of chromosome 13 (del(13q)) as the sole abnormality in chronic lymphocytic leukemia (CLL) portends a good prognosis; however, there is great outcome heterogeneity within this subgroup. The percentage of cells with a del(13q) (clone size) and the extent of the deletion are two factors that may affect outcome in CLL patients with isolated del(13q). We analyzed 248 CLL patients from the BC Provincial CLL database identified as having isolated del(13q) detected pretreatment by interphase fluorescence in situ hybridization to determine what impact clone and deletion size had on overall survival (OS) and treatment free survival (TFS). Patients with 60% or more of nuclei with a del(13q) had shorter TFS and shorter OS. A large deletion, encompassing the RB1 gene locus, was detected in half of the 90 cases with available specimens for testing, and there was no significant difference in OS and TFS between RB1-deleted and RB1-not-deleted cases. Further study in a larger sample size is required to determine the clinical interest of RB1 locus testing; however, clone size of del(13q) does predict TFS and OS and may better refine prognosis in this clinically heterogeneous population.

Exploring the somatic NF1 mutational spectrum associated with NF1 cutaneous neurofibromas

Neurofibromatosis type-1 (NF1), caused by heterozygous inactivation of the NF1 tumour suppressor gene, is associated with the development of benign and malignant peripheral nerve sheath tumours (MPNSTs). Although numerous germline NF1 mutations have been identified, relatively few somatic NF1 mutations have been described in neurofibromas. Here we have screened 109 cutaneous neurofibromas, excised from 46 unrelated NF1 patients, for somatic NF1 mutations. NF1 mutation screening (involving loss-of-heterozygosity (LOH) analysis, multiplex ligation-dependent probe amplification and DNA sequencing) identified 77 somatic NF1 point mutations, of which 53 were novel. LOH spanning the NF1 gene region was evident in 25 neurofibromas, but in contrast to previous data from MPNSTs, it was absent at the TP53, CDKN2A and RB1 gene loci. Analysis of DNA/RNA from neurofibroma-derived Schwann cell cultures revealed NF1 mutations in four tumours whose presence had been overlooked in the tumour DNA. Bioinformatics analysis suggested that four of seven novel somatic NF1 missense mutations (p.A330T, p.Q519P, p.A776T, p.S1463F) could be of functional/clinical significance. Functional analysis confirmed this prediction for p.S1463F, located within the GTPase-activating protein-related domain, as this mutation resulted in a 150-fold increase in activated GTP-bound Ras. Comparison of the relative frequencies of the different types of somatic NF1 mutation observed with those of their previously reported germline counterparts revealed significant (P=0.001) differences. Although non-identical somatic mutations involving either the same or adjacent nucleotides were identified in three pairs of tumours from the same patients (P<0.0002), no association was noted between the type of germline and somatic NF1 lesion within the same individual.

Altered structure and deregulated expression of the tumor suppressor gene retinoblastoma (RB1) in human brain tumors

A total of 40 human brain tumor samples were analyzed for tumor-specific alterations at the RB1 gene locus. Gliomas were more prevalent in younger males and meningiomas in older females. Southern blot analysis revealed loss of heterozygosity (LOH) at the intron 1 locus of RB1 gene in 19.4% of informative cases and this is the first report showing LOH at this locus in human brain tumors. Levels of RB1 mRNA and protein, pRb, and the percentage of hyperphosphorylated form of pRb were also analyzed in these tumors. Normal human fibroblast cell line WI38 was used as control in northern and western analysis. Normal sized RB1 mRNA and protein were present in all the tumor samples. Majority of the gliomas had 2.0-fold or higher levels of RB1 mRNA and most meningiomas had less than 2.0-fold of RB1 mRNA compared to control WI38 cells. The total pRb levels were 2.0-fold or higher in all the tumor samples compared to control. More than 50% of pRb existed in hyperphosphorylated form in all gliomas except two. However, six out of 13 meningiomas had less than 50% of total pRb in the hyperphosphorylated form. These results indicate that the increased percentage of hyperphosphorylated form of pRb in gliomas could provide growth advantage to these tumors. Presence of LOH at the RB1 gene locus and the increased levels of RB1 RNA and protein and increased percentage of hyperphosphorylated form of pRb are indicative of an overall deregulation of pRb pathway in human brain tumors.

Human retinoblastoma is not caused by known pRb‐inactivating human DNA tumor viruses

Retinoblastomas occur as the consequence of inactivation of the tumor suppressor retinoblastoma protein (pRb), classically upon biallelic inactivation of the RB1 gene locus. Recently, human papillomavirus (HPV) genomic DNA has been detected in retinoblastomas. To investigate the possibility that oncoproteins encoded by pRb-inactivating DNA tumor viruses play a role in the pathogenesis of human retinoblastoma, 40 fresh-frozen tumors were analyzed for the presence of HPV, adenovirus (HAdV) and polyomavirus (BKV, JCV and SV40) genomic DNA sequences by real-time polymerase chain reaction (PCR). Tumors were screened for genetic and epigenetic alterations in all 27 exons of the RB1 gene locus and promoter by exonic copy number detection, sequencing and methylation-specific PCR of the promoter region. Retinoblastoma tumors from children with bilateral familial (n=1), bilateral nonfamilial (n=1) and unilateral nonfamilial (n=38) disease were analyzed. Inactivating modifications to the RB1 gene locus were identified on both the alleles in 27 tumors, one allele in 8, and neither allele in 5 cases. A median of over 107,000 tumor cells were analyzed for viral genomic DNA in each PCR reaction. All tumor samples were negative for 37 HPV types, 51 HAdV types, BKV and JCV genomic sequences. Very low copy number (0.2-260 copies per 100,000 tumor cells) SV40 genomic DNA detected in 8 of 39 samples was demonstrated to be consistent with an artifact of plasmid-derived SV40. In contrast to recent reports, we obtained substantial quantitative evidence indicating that neither HPV nor any other pRb-inactivating human DNA tumor viruses play a role in the development of retinoblastoma, regardless of RB1 genotype.

Deletion mapping of chromosome 13q in head and neck squamous cell carcinoma in Indian patients: correlation with prognosis of the tumour

Deletions in chromosome (chr.) 13q occur frequently in head and neck squamous cell carcinoma (HNSCC). Previous studies failed to identify common deleted regions in chr.13q, though several candidate tumour suppressor genes (TSGs) loci, e.g. BRCA2, RB1 and BRCAX have been localized in this chromosome, as well as no prognostic significance of the deletion has been reported. Thus, in the present study, deletion mapping of chr. 13q has been done in 55 primary HNSCC samples of Indian patients using 11 highly polymorphic microsatellite markers of which three were intragenic to BRCA2 gene, one intragenic to RB1 gene and another from BRCAX locus. The deletion in chr.13q was significantly associated with progression of HNSCC. High frequencies (27-39%) of loss of heterozygosity were found in 13q13.1 (BRCA2), 13q14.2 (RB1), 13q21.2-22.1 (BRCAX) and 13q31.1 regions. Deletions in the BRCA2 and RB1 regions were significantly correlated. The four highly deleted regions were associated with clinical stage and histological grades of the tumour as well as poor patient outcome. Deletion in the 13q31.1 region was only found to be associated with HPV infection. High frequencies (11-23%) of microsatellite size alteration (MA) were seen to overlap with the highly deleted regions. Forty per cent of the samples showed rare biallelic alteration whereas loss of normal copy of chromosome 13q was seen in five tumours. Thus, it seems that the putative TSGs located in the BRCAX and 13q31.1 regions as well as the BRCA2 and RB1 genes may have some cumulative effect in progression and poor prognosis of HNSCC. Significant association between deletion in BRCA2 and RB1 gene loci may indicate functional relationship between the genes in this tumour progression.

Altered structure and expression of RB1 gene and increased phosphorylation of pRb in human vestibular schwannomas

Tumor-specific alterations at the RB1 gene locus in 30 human vestibular schwannomas including 10 NF2 and 20 sporadic cases were analysed. Southern blot analysis of DNA from these samples revealed loss of heterozygosity (LOH) at the RB1 locus in 6 of 24 informative cases (25%) compared to normal blood DNAs from the same patients. Northern blot analysis showed normal size RB1 mRNA in all the tumor samples. However, there was a 2-5-fold increase in the level of expression of the RB1 gene in all the tumor samples compared to the WI38 cell line which was used as control. Western blot analysis of the RB1 protein, pRb showed a 2.5-5-fold increase in the level of total pRb as compared to normal WI38 cell line. Sixty five to seventy five percent of the total pRb were in phosphorylated form in most tumors. The LOH at the RB1 gene locus suggests genetic instability in these patients. Further, increased levels of RB1 mRNA, total pRb and the phosphorylated form of pRb suggests that RB1 gene in these tumors may have anti-apoptotic function. These results suggest that the RB1 gene has a major role in the development of human vestibular schwannomas.

Clinical Use of Molecular Genetic Studies in Retinoblastoma

With a population incidence of about 1 in 15.000 retinoblastoma is the most frequent intraocular tumor in infancy and early childhood. It occurs in a hereditary form due to a germline mutation in about 40% of patients (30% de novo mutation and 10% transmission from an affected parent) and in a non-hereditary form due to a somatic mutation. The retinoblastoma gene is located on chromosome 13q14. This large gene of about 180 kb, consists of 27 exons of rather different sizes and encodes a 4.7 kb transcript with important function in cell cycle regulation. Individuals with bilateral, multifocal tumors are assumed to carry a germline mutation, whereas unilateral and unifocal tumors are generally due to the somatic form. Both copies of the RB1 gene must be in inactivated before a tumor develops. In about half of patients with the germline mutation the second event inactivating the second allele can be shown by loss of heterozygosity in tumor tissues compared to surrounding somatic tissues.Knowledge of the RB1 gene locus affords an opportunity to specify the type of mutation in many patients and arrive at a definitive molecular diagnosis. This is the basis for clinical evaluation and genetic counseling. The types of mutation are large scale deletions, small deletions and insertions, and base substitutions. There is no hot-spot for mutations.During the last several years we have studied more than 200 patients in search for large scale and small deletions and insertions, and missense mutations. Using intragenic polymorphic DNA markers we were able to identify the mutant haplotype in all familial cases. Direct DNA analysis identified a mutation in about 25% of patients. The distribution of lesions will be described in relation to the clinical situation.

Human pituitary adenomas show no loss of heterozygosity at the retinoblastoma gene locus.

The retinoblastoma tumor suppressor gene (RB1) is inactivated in hereditary and sporadic forms of retinoblastoma as well as in a number of other sporadic tumors. The majority of human pituitary tumors have been shown to be monoclonal neoplasms, suggesting that 1 or more somatic mutations are involved in the clonal expansion of a single progenitor cell. Recently, a high percentage of transgenic mice containing a disrupted RB1 allele have been shown to develop pituitary tumors. To investigate whether RB1 inactivation contributes to the development of human pituitary adenomas, we searched for loss of heterozygosity (LOH) within the RB1 gene locus in a variety of human pituitary adenomas. We screened 34 adenomas for LOH using a polymerase chain reaction (PCR)-based microsatellite polymorphic marker at the RB1 gene locus. In addition, a variable number of tandem repeat markers from within the RB1 gene was also used to search for LOH in 14 tumors. We found no LOH or microsatellite instability at the RB1 locus in any of the informative cases (30 of 34). Additionally, we showed that 4 representative adenomas from female patients are monoclonal in origin using a PCR-based clonality analysis assay. We conclude that the RB1 gene shows no LOH in a variety of human pituitary adenomas and that PCR-based microsatellite markers can serve as a useful tool for LOH analysis in human pituitary tumors.

Chromosome 13 instability and esterase D expression in an osteosarcoma cell line.

In order to assess the involvement of the 13q14 region in the development of osteosarcoma, both osteosarcoma tumor cells and normal tissue from a retinoblastoma patient previously used in restriction fragment length polymorphism studies, and sarcoma cells and normal fibroblasts from other tumor patients, have been investigated with respect to esterase D (E.C. 3.1.1.1) expression and chromosome pattern. In spite of an increased number of apparently normal chromosomes #13, a 50% reduction in esterase D activity in osteosarcoma cells from the retinoblastoma patient was observed. This suggests that loss of the RB1 gene or an OSRC gene closely linked to the ESD and RB1 gene loci is involved in the development of the osteosarcoma tumor. No reduction in esterase D expression was seen in four other sarcoma cell lines.