Comprehensive screening for constitutional RB1 mutations by DHPLC and QMPSF

Constitutional mutations of the RB1 gene are associated with a predisposition to retinoblastoma. It is essential to identify these mutations to provide appropriate genetic counseling in retinoblastoma patients, but this represents an extremely challenging task, as the vast majority of mutations are unique and spread over the entire coding sequence. Since 2001, we have implemented RB1 testing on a routine basis as part of the clinical management of retinoblastoma. As most screening techniques do not meet the requirements for efficient RB1 testing, we have devised a semi-automated denaturing high-performance liquid chromatography (DHPLC) method for point mutation detection combined with a quantitative multiplex PCR of short fluorescent fragments (QMPSF) approach to screen for gene rearrangements. We report the results of this comprehensive screening of all exons and promoter of RB1 in 192 unrelated patients, mostly of French origin. Among 102 bilateral and/or familial cases and 90 unilateral sporadic probands, mutations were identified in 83 (81.5%) and 5 (5.5%) cases, respectively. A total of 43 mutations have not been previously reported. The mutational spectrum was found to be significantly different from previous published series, displaying a surprising amount of splice mutations and large deletions. This study demonstrates the reliability of DHPLC for RB1 analysis, but also illustrates the need for a deletion scanning approach. Finally, considering the benefits to retinoblastoma patients, RB1 testing should be widely implemented in routine healthcare because our study clearly illustrates its feasibility.     

Spectrum of gross deletions and insertions in the RB1 gene in patients with retinoblastoma and association with phenotypic expression

Quantitative multiplex PCR and genomic real-time PCR were used to complete an RB1 mutation analysis in 57 of 433 and 72 of 262 patients with hereditary and isolated unilateral retinoblastoma, respectively. These patients were selected because in previous analyses, which focused mainly on the identification of point mutations, no RB1 mutation was found. We identified gross deletions and insertions in peripheral blood DNA from 26 of 57 patients (46%) with hereditary retinoblastoma, and in six of 72 patients (8.3%) with isolated unilateral disease. In addition, we identified 32 somatic mutations in tumor DNA from 31 of 72 patients (43%) with isolated unilateral retinoblastoma. Together with our previous results, we found that gross RB1 alterations were present in the peripheral blood DNA from 65 of 433 (15%) and 17 of 262 (6.5%) patients with bilateral or familial and isolated unilateral retinoblastoma, respectively. Including reported gross deletions, an analysis of the frequency of breakpoints per intron length shows higher densities in introns 13, 16, 23, and 24. Genotype-phenotype analyses showed that on the whole, carriers of gross deletions develop fewer retinoblastomas compared to patients who are heterozygous for other types of RB1 null mutations. Specifically, carriers of cytogenetic and submicroscopic whole gene deletions often have unilateral tumors only. By contrast, almost all patients with gross deletions with one breakpoint in RB1 have bilateral retinoblastoma.     

A molecular study of first and second RB1 mutational hits in retinoblastoma patients

RB1 mutations accountable for biallelic inactivation are crucial events in the development of retinoblastoma because a first mutation (M1) predisposes to retinoblastoma while a second mutation (M2) is required for tumor development. Mutational analyses of this gene showed a wide spectrum of genetic alterations (single base substitutions, insertions, or deletions, as well as small and large deletions). The most frequent second hit in retinoblastoma patients is loss of heterozygosity (LOH) followed by promoter methylation. Molecular analyses of RB1 mutations were conducted in 36 patients (20 unilateral and 16 bilateral) using polymerase chain reaction-mediated single-strand conformation polymorphism (SSCP) analysis, sequencing, and LOH analysis. Sixty-four amplified fragments showing abnormal SSCP patterns were sequenced, and mutations were confirmed in five patients (13.89%). Four mutations were located at coding regions, and a fifth one was found at an exon-intron junction. Two mutations were C-->T transitions, two were small-length deletions, and one was a G-->A transition. A total of 47.05% patients showed LOH. In one patient, the parental origin of the mutated allele was detected: the allele retained in the tumor was the paternal one. This work helps to characterize the spectrum of mutations in the Brazilian population, and to confirm that formaldehyde-fixed paraffin tissue can provide valuable information on the RB1 status in retinoblastoma patients.     

Enhanced Sensitivity for Detection of Low‐Level Germline Mosaic RB1 Mutations in Sporadic Retinoblastoma Cases Using Deep Semiconductor Sequencing

Sporadic retinoblastoma (RB) is caused by de novo mutations in the RB1 gene. Often, these mutations are present as mosaic mutations that cannot be detected by Sanger sequencing. Next‐generation deep sequencing allows unambiguous detection of the mosaic mutations in lymphocyte DNA. Deep sequencing of the RB1 gene on lymphocyte DNA from 20 bilateral and 70 unilateral RB cases was performed, where Sanger sequencing excluded the presence of mutations. The individual exons of the RB1 gene from each sample were amplified, pooled, ligated to barcoded adapters, and sequenced using semiconductor sequencing on an Ion Torrent Personal Genome Machine. Six low‐level mosaic mutations were identified in bilateral RB and four in unilateral RB cases. The incidence of low‐level mosaic mutation was estimated to be 30% and 6%, respectively, in sporadic bilateral and unilateral RB cases, previously classified as mutation negative. The frequency of point mutations detectable in lymphocyte DNA increased from 96% to 97% for bilateral RB and from 13% to 18% for unilateral RB. The use of deep sequencing technology increased the sensitivity of the detection of low‐level germline mosaic mutations in the RB1 gene. This finding has significant implications for improved clinical diagnosis, genetic counseling, surveillance, and management of RB.     

Twelve novel RB1 gene mutations in patients with hereditary retinoblastoma. Mutations in brief no. 206. Online

Hereditary predisposition to retinoblastoma is caused by germline mutations in the RB1 gene. Mutation analysis in this gene is important because knowledge of the causative mutation is often required for accurate risk prediction in relatives. We have performed RB1 gene mutation analysis in 45 patients with hereditary retinoblastoma. Screening by heteroduplex and SSCP analysis resulted in the identification of small mutations in 28 (62%) patients. Recurrent mutations, mostly CpG-transitions, were found in 16 patients. Two patients with isolated bilateral retinoblastoma showed missense mutations, S567L and C712R, which have previously been reported in a patient with bilateral tumors and in a family with low penetrance, respectively. Twelve of the mutations identified here have not been reported to date. These include a novel missense mutation, L662P, which was identified in two bilaterally affected siblings and their mother with unilateral retinoma.     

A microsatellite fluorescent method for linkage analysis in familial retinoblastoma and deletion detection at the RB1 locus in retinoblastoma and osteosarcoma.

Linkage analysis at the retinoblastoma locus (RB1) is essential for identifying individuals at risk and to offer adequate genetic counseling in familial retinoblastoma. It can also be used to detect large deletions involving RB1, which accounts for 15% of the genetic alterations in hereditary retinoblastoma. These studies are usually carried out with lengthy Southern blot analyses of relatively uninformative restriction fragment length polymorphisms. The authors report an alternative, reliable protocol for genotyping the RB1 locus using two pairs of highly informative intragenic and flanking microsatellites linked closely to the RB1 gene, and analysis of the fluorescent-labeled polymerase chain reaction products with automatic sizing technology. This methodology has successfully identified high risk carriers in five of the five pedigrees of familial retinoblastoma studied. In addition, gross deletions affecting the RB1 gene were identified in two of 12 sporadic bilateral retinoblastomas, and loss of heterozygosity at the RB1 locus has been detected in one of three osteosarcomas using the same experimental protocol. The described protocol is simpler and faster than conventional Southern blot methodologies and can identify a larger number of informative cases.     

pRB detection as a common event in human retinoblastomas: an immunohistochemical study

Approximately 30% of the cases of retinoblastoma (RB), the childhood eye cancer, are inherited and are manifested by unilateral or bilateral tumor. In sporadic tumors, accounting for 70% of cases, only one eye is affected. RB has three histological features: undifferentiated anaplastic cells, retinoblast pattern, and differentiated pattern characterized by Flexner Wintersteiner rosettes (FWR). Currently, results concerning phosphoprotein RB (pRB) expression in RB tumors are contradictory. In this study we detected pRB immunohistochemically in 10 tumors from bilateral or unilateral RBs, which did not show gross chromosomal alterations in cytogenetic studies. Interestingly, pRB was undetectable in only one tumor where we found distinct histological features. Our results suggest that pRB immunopositivity may be common in these tumors. However, it does not rule out the possibility that pRB is functionally inactive in some cases. This may be due to the protein being present in phosphorylated form or being altered by point mutations not affecting its expression. Another possibility is that mechanisms other than RB1 gene changes may lead to retinoblastoma because not all cases of retinoblastoma show gene alterations. Together these findings may be useful in understanding the molecular mechanisms associated with this type of pediatric tumor.     

Mutational screening of the RB1 gene in Indian patients with retinoblastoma reveals eight novel and several recurrent mutations

Retinoblastoma is the most common primary intraocular malignancy in children, caused by inactivation of the RB1 gene on chromosome 13. We carried out a mutational screen of the exons and promoter of the RB1 gene in Indian patients with retinoblastoma in order to determine the range of mutations giving rise to disease. Forty-seven patients were screened for mutations in all exons and promoter of the RB1 gene by single strand conformation polymorphism followed by sequencing. Tumors were available from 27 patients (12 bilateral and 15 unilateral retinoblastoma) while only peripheral blood was available from 20 patients, all with bilateral disease. Mutations were found in 22 patients, 9 from the analysis of tumors and 13 from peripheral blood. Eight novel mutations were identified, including 4 single base changes, 2 small deletions and 1 duplication. These are g.64365T>G (Tyr325Ter), g.78131G>A (Trp515Ter), g.150061G>T (Glu587Ter), g.170383C>G (S834X), g.41924A>C (IVS3-2A>C), g.150064ins4, g.160792del22, and g.76940del14 (IVS15 del +20-33). Almost all mutations produced nonsense codons or frameshifts. Recurrent mutations, especially at CpG sites were seen predominantly. Detectable mutations in exons were found in 46% of patients tested. Large deletions, epigenetic changes as well as mutations in non-coding regions may be the cause of disease in the remainder of patients. Knowledge of the full range of mutations can aid in the design of screening tests for individuals at risk.     

Loss of heterozygosity and mutations are the major mechanisms of RB1 gene inactivation in Chinese with sporadic retinoblastoma

We investigated sequence alternation, promoter methylation, and loss of heterozygosity (LOH) of the RB1 gene as possible mechanisms of its inactivation in retinoblastoma. In 42 Chinese patients with sporadic retinoblastoma, the promoter and entire coding region of RB1 were examined for sequence changes. Status of methylation of the CpG-rich island at the 5'end was determined by methylation specific PCR assay. We detected 15 RB1 mutations in 38% (16/42) of the retinoblastoma patients, among them 19% (8/42) were germ-line mutations. A total of nine novel mutations were identified: E54X, S114X, I126S, g73779insG, D718N, IVS2+1G>C, IVS14+1G>C, IVS21+1G>C, and a complex alteration g78177G>T/g78176insTT leading to 543X. Most of them are likely to affect the RB1large pocket domain through the production of truncated gene products. None of the DNA samples showed methylation at the RB1promoter. In 15 cases where both normal and cancerous retinoblastoma tissue specimens were available, allelic loss according to microsatellite markers within or distal to the RB1 locus was analyzed and immunohistological staining for RB1 expression performed. Among them, frequency of LOH at 13q14 was found to be high at 60% (9/15) with no segregation with unilateral tumors. All these nine tumors did not express RB1 protein, showing an association of LOH at the RB1 locus with its loss of expression in retinoblastoma. Our results indicate that the RB1 gene in sporadic retinoblastoma is commonly inactivated because of loss-of-function mutations and loss of heterozygosity but not by the epigenetic phenomenon of promoter hypermethylation.     

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.     

Spectrum of small length germline mutations in the RB1 gene

A screening method based on multiplexed automated fragment lengthanalysis of polymerase chain reaction products was used to identifygermline mutations in the RB1 gene. By screening 106 unrelatedpatients with hereditary retinoblastoma, 20 small deletions(1–18 bp) and seven insertions (1–5 bp) were Identified.When collating our data with reported mutations, recurrenceof small length mutations was observed at nine sites withinthe RB1 gene. Most of these contained mono-tonic runs or directrepeats embedded In homocopoly-mer tracts. While the majorityof mutations resulted In premature truncation, two mutationscaused an In-frame loss of F755 and G540 to E545, respectively.A genotype - phenotype comparison of patients carrying differentsmall length mutations did not reveal any consistent relation.Particularly, the two patients with In-frame mutations showeda high number of tumours consistent with regular-penetranceretinoblastoma.     

Constitutional genomic instability, chromosome aberrations in tumor cells and retinoblastoma

Although retinoblastoma (Rb) is initiated as a result of biallelic inactivation of the RB1 gene, additional genetic events (M3) in tumor cells are indicative of their role in the full transformation of retinal cells. We investigated the constitutional genetic instability by fragile site (FS) expression studies and checked its relationship with loci of tumor cytogenetics in a series of 36 retinoblastoma patients (34 nonfamilial and 2 familial cases). Tumor cytogenetics revealed -13/+13, del/t(13)(q14) (50%), +1/del/t(1p/q) (65%), +6/i(6p) (60%), and del(16)(q13)/(q22 approximately q23) (60%). Conventional cytogenetics in leukocytes revealed constitutional del(13q14) in five unilateral Rb (URB) and one trilateral Rb (TRB). Constitutional del(16)(q22) and t(6;12) were also identified in two cases. Constitutional FS analysis showed a significant increase in the cellular fragility, with high prevalence at 13q14, 3p14, 6p23, 16q22 approximately q23, and 13q22 loci in retinoblastoma patients (P<0.05). Patients with constitutional del(13)(q14) demonstrated higher fragility than those with normal constitution. A strong correlation between loci of constitutional FSs and loci of recurrent chromosomal abnormalities in tumors strengthen and support the proposal that FS loci present as inherent genomic instability in retinoblastoma. The chromosomal changes and resultant genetic mutations, along with RB1 mutation events, probably contribute synergistically to the development and progression of Rb malignancy. Implementation of fluorescence in situ hybridization to nonfamilial Rb on a large scale (113 cases) could detect constitutional RB1 deletion in 12.3% of cases, with equally higher incidence in URB (14.7%) and bilateral Rb (13.6%), demonstrating that the true prevalence of patients with predisposition to RB1 mutation in sporadic URB is definitely higher in our populations. Also, higher incidence of constitutional RB1 deletion mosaicism in unilateral than in bilateral Rb indicates that the constitutional genetic mosaicism in URB should be given serious consideration during genetic counseling.     

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.     

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.     

Next generation sequencing in sporadic retinoblastoma patients reveals somatic mosaicism

In about 50% of sporadic cases of retinoblastoma, no constitutive RB1 mutations are detected by conventional methods. However, recent research suggests that, at least in some of these cases, there is somatic mosaicism with respect to RB1 normal and mutant alleles. The increased availability of next generation sequencing improves our ability to detect the exact percentage of patients with mosaicism. Using this technology, we re-tested a series of 40 patients with sporadic retinoblastoma: 10 of them had been previously classified as constitutional heterozygotes, whereas in 30 no RB1 mutations had been found in lymphocytes. In 3 of these 30 patients, we have now identified low-level mosaic variants, varying in frequency between 8 and 24%. In 7 out of the 10 cases previously classified as heterozygous from testing blood cells, we were able to test additional tissues (ocular tissues, urine and/or oral mucosa): in three of them, next generation sequencing has revealed mosaicism. Present results thus confirm that a significant fraction (6/40; 15%) of sporadic retinoblastoma cases are due to postzygotic events and that deep sequencing is an efficient method to unambiguously distinguish mosaics. Re-testing of retinoblastoma patients through next generation sequencing can thus provide new information that may have important implications with respect to genetic counseling and family care.     

Molecular detection of constitutional deletions in patients with retinoblastoma

The recent cloning of the retinoblastoma (RB) gene as well as the identification of intragenic polymorphisms afford the necessary tools for the analysis of rearrangements using molecular hybridization. We searched for constitutional deletions by Southern blotting in 67 in dependent patients with normal karyotype comprising 15 familial and 52 sporadic cases. Among the latter, 33 were bilaterally and 19 unilaterally affected. We detected 6 deletions using cDNA probes covering almost all of the RB gene, as well as a genomic probe of the 5′ part of the gene. With this approach, the incidence of detectable deletions was around 10%. No hot spots for deletion breakpoints were found. Asymptomatic carriers were detected in 2 families. The effectiveness of genetic counselling was largely improved by this approach.