RB1 gene mutations in retinoblastoma.

Mutations in both alleles of the RB1 gene are causal for the development of retinoblastoma, a childhood tumor of the eye. The spectrum of somatic and germline mutations in this gene is dominated by small mutations. Data on small mutations are listed in a locus specific database available at http://www.d-lohmann.de/Rb/mutations.html. Analysis of 368 reported small mutations reveals considerable heterogeneity. A notable recurrence of transitions is observed at 13 CpG-dinucleotides that are part of CGA codons or splice donor sites. Most mutations create a premature termination codon. With few exceptions, patients heterozygous for mutations of this kind develop bilateral retinoblastoma. Missense mutations and inframe deletions are rare. Some of these mutations are associated with a distinct phenotype marked by incomplete penetrance and reduced expressivity.     

Detection of mosaic RB1 mutations in families with retinoblastoma

The RB1 gene mutation detection rate in 1,020 retinoblastoma families was increased by the use of highly sensitive allele specific‐PCR (AS‐PCR) to detect low‐level mosaicism for 11 recurrent RB1 CGA>TGA nonsense mutations. For bilaterally affected probands, AS‐PCR increased the RB1 mutation detection sensitivity from 92.6% to 94.8%. Both RB1 oncogenic changes were detected in 92.7% of sporadic unilateral tumors (357/385); 14.6% (52/357) of unilateral probands with both tumor mutations identified carried one of the tumor mutations in blood. Mosaicism was evident in 5.5% of bilateral probands (23 of 421), in 3.8% of unilateral probands (22 of 572), and in one unaffected mother of a unilateral proband. Half of the mosaic mutations were only detectable by AS‐PCR for the 11 recurrent CGA>TGA mutations, and not by standard sequencing. This suggests that significant numbers of low‐level mosaics with other classes of RB1 mutations remain unidentified by current technology. We show that the use of linkage analysis in a two‐generation retinoblastoma family resulted in the erroneous conclusion that a child carried the parental mutation, because the founder parent was mosaic for the RB1 mutation. Of 142 unaffected parental pairs tested, only one unaffected parent of a proband (0.7%) showed somatic mosaicism for the proband's mutation, in contrast to an overall 4.5% somatic mosaicism rate for retinoblastoma probands, suggesting that mosaicism for an RB1 mutation is highly likely to manifest as retinoblastoma. Hum Mutat 0, 1–10, 2009. © 2009 Wiley‐Liss, Inc.     

Identification of four novel RB1 germline mutations in Korean retinoblastoma patients

To elucidate RB1 germline mutations in Korean retinoblastoma patients, DNA samples from 14 children with bilateral (including three familial cases) and 19 children with unilateral retinoblastoma were analyzed. We found germline mutations in three out of 14 bilateral cases and one out of 19 unilateral cases. There were no germline mutations in the three familial cases. PCR-SSCP from each exon showed bandshifts in four patients which, upon sequencing, were shown to be K616E in exon 19 (c.1846A>G), an AA insertion in exon 7 (c.684-685insAA), R500G in exon 16 (c.1498A>G), and an A insertion in exon 23 (c.2391-2392insA), respectively. Hum Mutat 18:252, 2001.     

Point mutations in human GLI3 cause Greig syndrome

Greig cephalopolysyndactyly syndrome (GCPS, MIM 175700) is a rare autosomal dominant developmental disorder characterized by craniofacial abnormalities and post-axial and pre-axial polydactyly as well as syndactyly of hands and feet. Human GLI3, located on chromosome 7p13, is a candidate gene for the syndrome because it is interrupted by translocation breakpoints associated with GCPS. Since hemizygosity of 7p13 resulting in complete loss of one copy of GLI3 causes GCPS as well, haploinsufficiency of this gene was implicated as a mechanism to cause this developmental malformation. To determine if point mutations within GLI3 could be responsible for GCPS we describe the genomic sequences at the boundaries of the 15 exons and primer pair sequences for mutation analysis with polymerase chain reaction-based assays of the entire GLI3 coding sequences. In two GCPS cases, both of which did not exhibit obvious cytogenetic rearrangements, point mutations were identified in different domains of the protein, showing for the first time that Greig syndrome can be caused by GLI3 point mutations. In one case a nonsense mutation in exon X generates a stop codon truncating the protein in the C-H link of the first zinc finger. In the second case a missense mutation in exon XIV causes a Pro-->Ser replacement at a position that is conserved among GLI genes from several species altering a potential phosphorylation site.      

Identification of novel mutations in the RB1 gene in Mexican patients with retinoblastoma

Retinoblastoma (RB) is a childhood tumor of the eye with an average incidence of one case in every 15,000-20,000 live births and occurs in sporadic or hereditary form. This cancer results from loss or inactivation of the RB1 gene located at 13q14.1. This gene encodes for a 110 Kd nuclear phosphoprotein (pRB) that plays a major role in cell proliferation control. Different types of mutations in the RB1 gene have been reported, but point mutations are the most common. There are no molecular studies on RB1 gene mutation in Mexican patients. In this study, 19 patients with bilateral or unilateral RB were analyzed. Genetic and cytogenetic studies were carried out. Detection of RB1 gene mutations was done using single-strand conformational polymorphism (SSCP). Five conformational polymorphisms were identified in different exons. In all cases, SSCP sequence showed new non-described mutations that produced a frameshift on the open reading frame. The identification of mutations in the RB1 gene contributes to basic knowledge of this neoplasia and permits the possibility to offer adequate genetic counseling to relatives at risk.     

miR-17~92 cooperates with RB pathway mutations to promote retinoblastoma

The miR-17~92 cluster is a potent microRNA-encoding oncogene. Here, we show that miR-17~92 synergizes with loss of Rb family members to promote retinoblastoma. We observed miR-17~92 genomic amplifications in murine retinoblastoma and high expression of miR-17~92 in human retinoblastoma. While miR-17~92 was dispensable for mouse retinal development, miR-17~92 overexpression, together with deletion of Rb and p107, led to rapid emergence of retinoblastoma with frequent metastasis to the brain. miR-17~92 oncogenic function in retinoblastoma was not mediated by a miR-19/PTEN axis toward apoptosis suppression, as found in lymphoma/leukemia models. Instead, miR-17~92 increased the proliferative capacity of Rb/p107-deficient retinal cells. We found that deletion of Rb family members led to compensatory up-regulation of the cyclin-dependent kinase inhibitor p21Cip1. miR-17~92 overexpression counteracted p21Cip1 up-regulation, promoted proliferation, and drove retinoblastoma formation. These results demonstrate that the oncogenic determinants of miR-17~92 are context-specific and provide new insights into miR-17~92 function as an RB-collaborating gene in cancer.     

Oncogenic point mutations in the human retinoblastoma gene: their application to genetic counseling

Mutations of the retinoblastoma gene, most of which cannot be detected by conventional Southern blotting, are known to cause both the nonhereditary and hereditary forms of retinoblastoma and have been implicated in the development of other cancers. Nonhereditary retinoblastoma is caused by a somatic mutation. Hereditary retinoblastoma is caused by a germ-cell mutation, most often a new one, and thus there is usually no family history of the disease. Unlike patients with the nonhereditary disease, those with the hereditary form are at risk for additional retinoblastomas, and their progeny are at risk for the tumors. We used a sensitive technique of primer-directed enzymatic amplification, followed by DNA sequence analysis, to identify mutations as small as a single nucleotide change in tumors from seven patients with simplex retinoblastoma (with no family history of the disease). In four patients the mutation involved only the tumor cells, and in three it involved normal somatic cells as well as tumor cells but was not found in either parent; thus, these mutations appeared to be new, germ-cell mutations. In addition, we found point mutations in cells from a bladder carcinoma, a small-cell carcinoma of the lung, and another retinoblastoma. We conclude that the technique that we have described can distinguish hereditary from nonhereditary retinoblastoma and that it is useful in risk estimation and genetic counseling.     

Two independent RB1-inactivating mutations in peripheral blood DNA of a hereditary retinoblastoma patient

We report the presence of a hemizygous inactivating germ-line RB1 mutation (a recurrent g.78250C-->T transition, resulting in a stop codon in exon 17) in peripheral blood DNA from a patient with hereditary bilateral retinoblastoma. Hemizygosity was established by sequencing that showed no traces of the wild-type C nucleotide and by quantitative real-time PCR, which showed loss of one copy of exon 17. Genotyping of the RB1 locus with several polymorphic markers delineated a maximal deletion region between g.76875 and g.99426, including exons 15-17 and a large piece (21 kb) of intron 17. The heterozygosity for the mutation found in skin fibroblasts proves that the intragenic RB1 deletion probably took place in the definitive hematopoietic lineage of the patient. The presence of a null Rb-/- genotype in the hematopoietic cell lineage suggests that the white blood cells of the proband could be useful in the investigation of the role of complementary RBI family proteins in the control of the cell cycle.     

Bilateral retinoblastoma with ectopic intracranial retinoblastoma: Trilateral retinoblastoma

In 11 patients, bilateral retinoblastoma presented at a mean age of 6 months and pineoblastoma at 4 years. We suggest that the hereditary multicentric retinoblastoma arose in vestigeal photoreceptors in the pineal as well as in the hypothetical retinoblasts of the retina. In certain lower animals, the pineal functions as a photoreceptor organ, resembles the retina histologically, and is described as a “third eye”. Hence, the patients we describe may be considered as having “trilateral retinoblastoma”. Two possible variants of this entity were also noted: (1) three children without retinoblastoma who developed pineoblastoma with rosettes and photoreceptor differentiation characteristic of retinoblastoma, and (2) three additional cases involving children who presented with retinoblastoma-like tumors in the suprasellar or parasellar region 2 to 6 months before the discovery of intraocular retinoblastoma. These observations suggest that the retinoblastoma gene confers a previously unappreciated susceptibility to a narrow spectrum of neuroblastic tumors, which usually present in the retina but which can also occur ectopically.     

New RB1 oncogenic mutations and intronic polymorphisms in Serbian retinoblastoma patients: genetic counseling implications

The purpose of this work was to identify germ line RB1 mutations in 16 Serbian retinoblastoma patients for genetic counselling. Mutation analysis was carried out by PCR directed sequencing of the 27 exons. Loss of heterozygosity for two RB1 intragenic markers was also analyzed in 14 tumour samples. Five new RB1 oncogenic mutations (g.2078 del C, g.77047_48 del GC, g.78117_8 del TT, g.160797 del T, and g.64439+2 T>C) and two recurrences (R445X and Q383X) have been found in this study. In addition, four intronic variants were observed germ line in some unilateral patients. Two of these variants (g.44668-15T/G, and g.166204-8T/A) are discussed as potential oncogenic mutation candidates. The results show the relevance of studies aimed to investigate the role of intronic variants in exon splicing regulation. Such studies will help to disclose hidden retinoblastoma susceptibilities, important for accurate genetic counselling.     

Mutational screening of the RB1 gene in Italian patients with retinoblastoma reveals 11 novel mutations

Retinoblastoma (RB, OMIM#180200) is the most common intraocular tumour in infancy and early childhood. Constituent mutations in the RB1 gene predispose individuals to RB development. We performed a mutational screening of the RB1 gene in Italian patients affected by RB referred to the Medical Genetics of the University of Siena. In 35 unrelated patients, we identified germline RB1 mutations in 6 out of 9 familial cases (66%) and in 7 out of 26 with no family history of RB (27%). Using the single-strand conformational polymorphism (SSCP) technique, 11 novel mutations were detected, including 3 nonsense, 5 frameshift and 4 splice-site mutations. Only two of these mutations (1 splice site and 1 missense) were previously reported. The mutation spectrum reflects the published literature, encompassing predominately nonsense or frameshift and splicing mutations. RB1 germline mutation was detected in 37% of our cases. Gross rearrangements outside the investigated region, altered DNA methylation, or mutations in non-coding regions, may be the cause of disease in the remainder of the patients. Some cases, e.g. a case of incomplete penetrance, or variable expressivity ranging from retinoma to multiple tumours, are discussed in detail. In addition, a case of pre-conception genetic counselling resolved by rescue of banked cordonal blood of the affected deceased child is described.     

Spectrum of germline RB1 gene mutations in Spanish retinoblastoma patients: Phenotypic and molecular epidemiological implications

Mutation analysis of retinoblastoma is considered important for genetic counseling purposes, as well as for understanding the molecular mechanisms leading to tumors with different degrees of penetrance or expressivity. In the course of an analysis of 43 hereditary retinoblastoma Spanish patients and kindred, using direct PCR sequencing, we have observed 29 mutations; most of them (62%) have not been reported previously. Of the mutations, 69% correspond to nonsense mutations (mainly CpG transitions) and frameshifts, with the expected outcome of a truncated Rb protein that lacks the functional pocket domains and tail. The remainder corresponds to splicing mutations, most of them (62%) targeted to invariant nucleotides, with the predicted consequence of out of frame exon skipping. Two of the splicing mutations in our study were found associated to families with a low-penetrance phenotype. Additionally, most of the mutations affecting splice junctions corresponded to retinoblastoma cases of either sporadic or hereditary nature with delayed onset (32 months on average). In contrast, most of the nonsense and frameshift mutations are associated with an early age at diagnosis (8.7 months on average). These differences are discussed in the context of the relationships between genotype and low expressivity phenotype. The differences in the spectrum of RB1 mutations found in this and other European surveys are also discussed in the context of alternate DNA methylation and mismatch repair phenotypes.     

Glycoconjugates in retinoblastoma

Summary The binding of eleven biotin- or peroxidase-coupled lectins with different carbohydrate specificities to tumour tissue and remaining morphologically normal retina was studied in ten formalin-fixed and paraffin-embedded human eyes with retinoblastoma. In undetached retinas, outer and inner segments of photoreceptors bound concanavalin A (ConA) as well asLens culinaris (LCA), wheat germ (WGA)Ricinus communis (RCAI) and peanut (PNA) agglutinins. Both nuclear and plexiform layers bound ConA, LCA and, in some specimens, WGA and RCAI. These results agree with those obtained with normal adult human retina, the main difference being that PNA labelled some rods in addition to cones in the retinoblastoma eyes. Flexner-Wintersteiner rosettes reacted with ConA and LCA, and often with WGA, PNA and RCAI. Undifferentiated retinoblastoma cells always bound ConA and LCA, and in some tumours WGA, PNA and RCAI. Pretreatment with neuraminidase increased the number of cells that bound PNA and RCAI, but diminished binding of WGA. Pokeweed mitogen andBandeiraea simplicifolia I, Dolichos biflorus, soybean,Ulex europaeus I andLotus tetragonolobus agglutinins labelled only vascular endothelial cells. Retinoblastoma cells most closely resembled photoreceptor cells in their lectin-binding patterns.This study was supported by grants from the Emil Aaltonen Foundation and Rasmussens Stiftelse     

Identification of 26 new constitutional RB1 gene mutations in Spanish, Colombian, and Cuban retinoblastoma patients

Constitutional mutations in the RB1 gene predispose to retinoblastoma development. Hence genetic screening of retinoblastoma patients and relatives is important for genetic counseling purposes. In addition, RB1 gene mutation studies may help decipher the molecular mechanisms leading to tumors with different degrees of penetrance or expressivity. In the course of genetically screening of 107 hereditary and non-hereditary retinoblastoma patients (11 familiar bilateral, 4 familiar unilateral, 49 sporadic bilateral and 43 sporadic unilateral) and kindred from Spain, Colombia and Cuba, using direct PCR sequencing, we observed 45 distinct mutations and four RB1 deletions in 53 patients (9 familiar bilateral, 2 familiar unilateral, 31 sporadic bilateral and 11 sporadic unilateral). Most of these mutations (26/45, 57%) have not been reported before. In 32 patients, the predisposing mutations correspond to nonsense (mainly CpG transitions) and small insertions or deletions whose expected outcome is a truncated Rb protein that lacks the functional pockets and tail. Five single aminoacid replacements and seventeen mutations affecting splicing sites were also observed in retinoblastoma patients. Two of these sixteen mutations are of unclear pathogenic nature.     

Chromosomes in retinoblastoma patients

In a series of eight patients with retinoblastoma, one was found to have a reciprocal translocation of chromosomes 1 and 13. The breakpoint on chromosome 13 is at band q12, which suggests that the retinoblastoma locus is less distal than previously thought.     

Point mutations in the AML1/RUNX1 gene associated with myelodysplastic syndrome

Myelodysplastic syndrome (MDS) is a clonal disorder of hematopoietic stem cells characterized by ineffective and inadequate hematopoiesis. Because MDS is a heterogeneous disorder, specific gene abnormalities implicated in the pathogenesis of MDS have been difficult to identify. Cytogenetic abnormalities are seen in half of the MDS patients and generally consist of partial or complete chromosome deletion or addition, whereas balanced translocations are rare. Although point mutations of critical genes had been demonstrated to contribute to the development of MDS, there was no strong correlation between these mutations and clinical features. Recently, we reported the high incidence of somatic mutations in the AML1/RUNX1 gene (which is a critical regulator of definitive hematopoiesis and the most frequent target for translocation of acute myeloid leukemia [AML]) in MDS, especially refractory anemia with excess blasts (RAEB), RAEB in transformation (RAEBt), and AML following MDS (defined here as MDS/AML). The MDS/AML patients with AML1 mutations had a significantly worse prognosis than those without AML1 mutations. Most AML1 mutants lose trans-activation potential, which leads to a loss of AML1 function. These data indicate that AML1 point mutation is one of the major causes of MDS/AML, and "MDS/AML with AML1 mutation" represents a distinct clinicopathologic-genetic entity.