Tumor Biol. DOI 10.1007/s13277-014-2851-7
RESEARCH ARTICLE
Novel mutations in the RB1 gene from Chinese families with a history of retinoblastoma Leilei Zhang & Renbing Jia & Junyang Zhao & Jiayan Fan & YiXiong Zhou & Bing Han & Xin Song & Li Wu & He Zhang & Huaidong Song & Shengfang Ge & Xianqun Fan
Received: 12 August 2014 / Accepted: 13 November 2014 # International Society of Oncology and BioMarkers (ISOBM) 2014
Abstract Retinoblastoma is an aggressive eye cancer that develops during infancy and is divided into two clinical types, sporadic and heritable. RB1 has been identified as the only pathological gene responsible for heritable retinoblastoma. Here, we identified 11 RB1 germline mutations in the Han pedigrees of 17 bilateral retinoblastoma patients from China. Four mutations were nonsense mutations, five were splice site mutations, and two resulted in a frame shift due to an insertion or a deletion. Three of the mutations had not been previously reported, and the p.Q344L mutation occurred in two generations of retinoblastoma patients. We investigated phenotypic– genotypic relationships for the novel mutations and showed that these mutations affected the expression, location, and function of the retinoblastoma protein. Abnormal protein Leilei Zhang, Renbing Jia, and Junyang Zhao contributed equally to this article. L. Zhang : R. Jia : J. Fan : Y. Zhou : X. Song : H. Zhang : S. Ge (*) : X. Fan (*) Department of Ophthalmology, Shanghai Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine, No. 639 Zhi Zao Ju Road, Shanghai 200011, People’s Republic of China e-mail: [email protected] e-mail: [email protected] J. Zhao Beijing Tongren Eye Centre, Beijing Ophthalmology & Visual Sciences Key Lab, Tongren Hospital, Capital Medical University, Beijing Economic-Technological Development Area, Beijing 100176, People’s Republic of China B. Han : H. Song Department of Endocrinology, Shanghai Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine, No. 639 Zhi Zao Ju Road, Shanghai 200011, People’s Republic of China L. Wu Department of Ophthalmology, People’s Hospital, Wu Han University, No. 238 Jie Fang Road, Wu Han 430060, People’s Republic of China
localization was observed after transfection of the mutant genes. In addition, changes in the cell cycle distribution and apoptosis rates were observed when the Saos-2 cell line was transfected with plasmids encoding the mutant RB1 genes. Our findings expand the spectrum of known RB1 mutations and will benefit the investigation of RB1 mutation hotspots. Genetic counseling can be offered to families with heritable RB1 mutations. Keywords Retinoblastoma . RB1 . Mutation . Genetic counseling
Introduction Retinoblastoma [MIM 180200; RB] [1] is the most common intraocular malignancy in children, with an incidence ranging from 1 in 15,000 to 1 in 20,000 live births [2]. There is no racial or gender predisposition for the development of RB, which is currently split into two clinical types: sporadic and heritable. The average age at the time of diagnosis is 23 months, with unilateral cases being diagnosed at approximately 27 months and bilateral cases at 15 months [3]. All bilateral RB cases are heritable, but only a small proportion of unilateral cases can be passed on to future generations [4]. Hereditary RB, which accounts for 40 % of cases, is transmitted as an autosomal dominant trait that presents as bilateral disease, due to high expressivity (90 %). In 1971, Knudson proposed the “two-hit hypothesis,” which was confirmed in 1986 by the discovery of the RB1 [NM_000321] gene in region 14 of chromosome 13 by Dryja et al. [5]. One allele is mutated in the germline and predisposes the child to retinal tumors, while the second allele is mutated at the cellular level and initiates tumor progression. However, in the nonhereditary disease, both alleles are mutated at the cellular level. RB metastases are rare in the developed world, where early
Tumor Biol.
diagnosis can result in a cure for most children. RB confined to the eye can be cured through simple enucleation. However, curing RB that has metastasized out of the eye is rare [6]. The RB1 gene, located on chromosome 13q14, was the first tumor suppressor gene to be identified. The RB1 gene contains 27 exons distributed over 200 kb of DNA [7] and encodes the retinoblastoma protein [RB protein; NP_000312]. Knowledge of RB1 gene mutations is important for genetic counseling and the characterization of phenotypic–genotypic relationships. To date, more than 900 RB1 germline mutations have been published, and nearly 40 % of RB1 mutations are recurrent [8] and frequently cause gene inactivation. Despite missense mutations being the main inactivating events in most genetic diseases, deletions and nonsense mutations are more often responsible for RB1 inactivation. In 2005, a review indicated that the nonsense mutation rate had reached 42 % [8]. Approximately 60 % of RB1 nonintermediary germline mutations can result in sporadic cases of unilateral disease, with no family history of RB. These RB1 mutations arise locally within the developing retina [9]. However, once an RB1 mutation is present in the germline, it can result in the hereditary transmission of this disease. Individuals with hereditary RB have a 50 % chance of passing these mutations on to their offspring. An RB1 mutation also results in an increased risk of secondary malignancies in the lung, bladder, bone, soft tissues, skin, and brain throughout the patient’s life, particularly when the children are treated with radiation [1]. These secondary, nonocular malignancies are more lethal than RB. In this study, we performed genetic screening of Chinese RB patients and their family members for heritable RB1 mutations. Direct sequencing was used to analyze the RB1 gene and explore potential mutation hotspots. We also investigated the phenotypic–genotypic relationships of two novel mutations in human Saos-2 cells. To the best of our knowledge, this is the first description of these novel mutations.
Materials and methods Subjects and DNA extraction All DNA samples were obtained from the Department of Shanghai Ninth Hospital, and genetic counseling was provided to all participants. Informed consent was obtained from each family member prior to participation in the study, under the authority of the Institutional Ethics Committee of Shanghai Ninth Hospital. Fresh venous blood samples were collected from the patients and their family members and 100 individuals with neither a personal nor family history of RB. The 100 nonRB samples were included as controls to exclude the possibility that the mutations were common polymorphisms. All control samples were from Han Chinese people having the same genetic background. Whole blood was
collected in EDTA tubes. Genomic DNA was extracted from peripheral blood leukocytes (5 ml) using the Automatic Nucleic Acid Isolation System (QuickGene-610L, Fujifilm Life Science, Tokyo, Japan). PCR and sequencing Screening for RB1 mutations was performed through direct polymerase chain reaction (PCR) sequencing of the 27 coding exons and their flanking intron regions. The primer sets and PCR annealing temperatures are described in Table 3. PCR reactions were performed in a thermal cycler in a total volume of 20 μl containing 100 ng of genomic DNA, 5 pmol of each primer, and 7.5 μl of 2× Master Mix (AmpliTaq Gold 360 Master Mix; Applied Biosystems, Foster City, CA, USA). The PCR cycling conditions included an initial denaturation at 95 °C for 5 min followed by 35 cycles of denaturation at 94 °C for 1 min, annealing at the specific temperature for 1 min, 72 °C for 1 min, followed by a final extension step of 72 °C for 7 min. The presence of a specific PCR product was confirmed using 1.5 % agarose gel electrophoresis. Reactions that displayed single bands, without nonspecific amplification, were chosen for sequencing. The mutations were confirmed by sequencing in both the forward and reverse directions on an ABI 3730XL DNA sequencer (Applied Biosystems PerkinElmer). In some cases, a second amplification was required to confirm the mutation. Molecular modeling RB1 mutations can lead to an amino acid substitution in RB protein. To predict the possible impact of a substitution on the structure and function of RB protein, a three-dimensional computer model was used to analyze the structural positioning of the mutation sites. The crystal structure of the DNAbinding domain of RB protein was obtained from the Protein Data Bank (PDB) (code, 4ELJ, chain A). The DNAbinding domain of RB protein was modeled on the basis of homology using SWISS-MODEL, as previously described [10]. Construction of the expression vectors Professor Zhao (Southern Medical University, Key Laboratory for Proteomics of in Guangdong Province) provided a plasmid that contained the wild-type RB1 gene. The wild-type RB1 gene was attached to an enhanced green fluorescent protein (EGFP) tag, and the wild-type RB1 and wild-type RB1-EGFP plasmids were used as template to construct the mutant plasmids. The mutations without the EGFP tag were used for fluorescein isothiocyanate (FITC)/Annexin V apoptosis staining cell cycle distribution analysis, while the mutants with the
Tumor Biol.
EGFP tag were used for subcellular location analysis. The mutant expression vectors were generated using site-specific mutagenesis, with the wild-type plasmids as templates. The following primers were used for p.D286X: sense 5′-CATGAA TGTAATATATAGATGAGGTG-3′ and antisense 5′-TATATA TTACATTCATGTTCTTTACAGAG-3′. The following primers for p.Q344L were used: sense 5′-ATGATAAAACTC TTCTGATTCTATAGACAG-3′ and antisense 5′-GAAGAG TTTTATCATGATCCAAAAA-3′. The reactions were performed using a touchdown PCR protocol with an initial denaturation of 95 °C for 5 min followed by 16 cycles of denaturation at 94 °C for 50 s, annealing at 64 °C for 1 min, and elongation at 68 °C for 11 min. After each cycle, the annealing temperature was decreased by 0.5 °C. An additional 16 cycles of denaturation at 94 °C for 50 s, annealing at 56 °C for 1 min, and extension at 68 °C for 11 min were performed, followed by a final extension step at 68 °C for 11 min. The KOD-Plus-Neo enzyme (TOYOBO, Osaka, Japan) was used to perform the touchdown PCR reactions. After completion of the PCR reaction, the wild-type plasmid was removed with the Dpn1 enzyme (Sangon, Shanghai, China) which only targets methylated sites within the plasmid that are not present in the PCR products. The mutated plasmids were isolated and purified using anion exchange columns (TIANGEN BIOTECH, Beijing, China). All constructs were sequenced and confirmed without single mutations at other sites. Cell culture and transfection The Saos-2 cell line was kindly provided by Professor Hao (Shanghai, China). The 293T cell line was purchased from the American Type Culture Collection (Manassas, VA, USA). Both Saos-2 and 293T cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, CA, USA), supplemented with 10 % fetal bovine serum (FBS; Gibco– Invitrogen, Grand Island, NY, USA) in a 5 % CO2 atmosphere. Transfection was performed using Lipofectamine 2000 (Invitrogen, CA, USA), according to the manufacturer’s instructions. A day before transfection, 2×105 cells were plated in 2 ml of growth medium, without antibiotics, to ensure that the cells were 50–80 % confluent at the time of transfection. Transfection was performed the following day; afterwards, the serum-containing culture medium was changed to serum-free culture medium. Four hours after transfection, the serum-free culture medium was replaced to serumsupplemented culture medium. Cell cycle analysis Wild-type and mutant plasmids were transfected into Saos-2 cells, which were harvested 48 h after transfection, along with control cells that had not undergone transfection. The cells were washed twice with cold phosphate-buffered saline
(PBS), fixed in 70 % ethanol, and stored at 4 °C overnight. On the next day, they were then washed twice with cold PBS and incubated with propidium iodide/ribonuclease staining solution (Becton Dickinson, NJ, USA) for 15 min at room temperature, following the manufacturer’s instructions. Cell cycle distribution was detected and analyzed using the FACScan instrument and CellQuest program (Becton Dickinson, NJ, USA). Apoptosis analysis Forty-eight hours after transfection, the cells were harvested and washed in cold PBS. Apoptosis was determined through flow cytometry using the FITC/Annexin V Apoptosis Detection Kit (Becton Dickinson, NJ, USA), according to the manufacturer’s instructions. Samples were examined by the FACScan instrument (Becton Dickinson, NJ, USA), and data were analyzed using the CellQuest programs (Becton Dickinson, NJ, USA). Immunofluorescence 293T cells transfected with plasmids encoding wild-type RB1 or the p.D286X and p.Q344L RB1 mutants were seeded onto glass coverslips (VWR, West Chester, PA, USA) in 12-well plates and cultured overnight. Forty-eight hours after transfection, cells were fixed in 4 % (w/v) paraformaldehyde (SigmaAldrich, St. Louis, MO, USA) for 15 min at room temperature. The coverslips were washed again, and the nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI; Vector Laboratories, Burlingame, CA, USA) for 2 min at room temperature. The control samples were processed using the same protocol, but the primary antibody was omitted. Immunoreactive cells were visualized using a fluorescent microscope (Olympus BX51, Japan). Western blot (immunoblot) analysis The protein extracted from cells and supernatants was collected by centrifugation at 12,000×g at 4 °C for 30 min. The total protein concentration was determined using the BCA protein assay kit (Pierce, Rockford, IL, USA). Equal amounts of total cellular extracts (20 μg) were separated by SDS-PAGE and blotted onto PVDF membranes (Millipore, USA). After the addition of blocking solution (5 % nonfat dry milk/0.1 % Tween-20/PBS), the membranes were incubated overnight with the recommended dilution of the following primary antibodies: anti-RB1 (1:1,000, Epitomics, USA); anti-activatedcaspase-3 (1:1,000, caspase-3 p17, Bioworld Technology, USA); and anti-ß-actin (1:10,000, Invitrogen, USA). After incubation with the primary antibodies at 4 °C overnight, the membranes were washed three times with TBS containing 0.1 % Tween-20 (TBST) and incubated with a secondary
Tumor Biol.
antibody (Invitrogen, USA) conjugated to a fluorescent tag for 1 h. The membranes were then washed three times with TBST. The bands were detected using the Odyssey infrared imaging system (Odyssey; LI-COR, Lincoln, NE, USA). Cell viability Cell viability was quantified using cell counting kit-8 (CCK8, Dojindo, Kumamoto, Japan) according to the manufacturer’s instruction. In brief, Saos-2 cells were plated to 96-well plates at a concentration of 2,000 cells/well with five duplicates. After overnight, the cells were transfected with wild-type RB1 and mutants. Four hours later, the medium was changed to DMEM with 10 % FBS and then treated for 24, 48, and 72 h. Absorbance was measured after a further 3-h incubation at 37 °C with an addition of cell counting kit-8 (10 μg/ml). The absorbance was measured on a spectrophotometer microplate reader at a wavelength of 450 nm.
Results Genetic analysis DNA from the RB patients was analyzed using reverse transcription PCR and was sequenced to obtain the complete coding sequence of the RB1 gene. Seventeen RB patients were analyzed, and 11 mutations were detected. Two mutations were novel with one recurred twice and nine had been reported previously (Table 1). Five mutations were single base substitutions (p.Q762X, p.D286X, p.R255X (observed twice), and p.G125X) that led to nonsense amino acid changes (Fig. 1a). The previously reported splice site mutations
Table 1
Fig. 1 A summary of the RB1 gene mutations in RB patients. a Schematics show the human wild-type and mutant versions of RB1. The N-terminal domain is shown in blue. The yellow portion of the diagram indicates the pocket-like structures of RB protein, which extends from residue 379 to 792 and plays an important role in RB1 function. The mutants are shown below including p.Q762X, p.Q344L, p.D286X, p.R255X, and p.G125X, with the C-terminal domain removed. With the exception of p.Q762X, the pocket-like domain of other mutants is removed. b–d The sequences of the p.G125X, p.R255X, and p.Q762X mutations. e–h, i–l, and m–p The two structure types of the mutants and the corresponding wild-type sequences. e, g, i, k, m, and o The space filling conformations. f, h, j, l, n, and p The ribbon conformation, e, f protein structure of the p.G125X mutant. i, g protein structures of p.R255X, and m, n molecular modeling of p.Q762X, all of which lead to the premature termination of the protein. g, h, k, l, o, and p Corresponding wild-type protein structures of p.G125X, p.R255X and p.Q762X, respectively, which are shown without deletion of the partial protein structure (indicated by white arrows)
(IVS22 as −2 A-G) were observed five times in different patients in this study. One novel mutation led to the deletion of four base pairs, AGAC (c.1031_1034del4nt, p.Q344L), in the open reading frame of a male patient. The family history of this patient was positive for RB, and the same mutation was present in the boy’s father, which is an inherited mutations. The pedigree of this family is shown in Fig. 2a. This variant was not observed in 100 control individuals or the patient’s mother. This result indicated that p.Q344L may play an important role in the initiation and progression of RB in this family. In addition, several databases, including NCBI, dbSNP, and UCSC, were searched to ensure that this substitution was not a single-nucleotide polymorphism. Our findings indicated that this was indeed a novel mutation that was closely associated with RB in this Chinese family. The p.D286X (c.856-857insert2nt) mutation was also novel and was found in exon 8 of a bilateral RB patient with no family history of RB (Fig. 2b), which inserted two nucleotides (TA).
RB1 germline mutations identified in Chinese patients with retinoblastoma
No.
RB phenotype
Type of mutation
cDNA
Protein
Exon location
Functional consequences
Reported
1a-10 1a-23
Bilateral Bilateral
Deletion Splicing
c.1031_1034del4nt IVS22 as −2 A-G
p.Q344 L
10 23
Gln-Leu
No Yes [11]
2a-3 2a-23 3a-23 5a-23 6a-23 3a-8 12a-8 4a-22 5a-8
Bilateral Bilateral Bilateral Bilateral Bilateral Bilateral Bilateral Bilateral Bilateral
Nonsense Splicing Splicing Splicing Splicing Substitution Substitution Nonsense Insertion
c.373 G>T IVS22 as −2 A-G IVS22 as −2 A-G IVS22 as −2 A-G IVS22 as −2 A-G c.763 C>T c.763 C>T c.2284 C>T c.856-857insert2nt
p.G125X
3 23 23 23 23 8 8 22 8
Glu-Term
Yes [12] Yes [11] Yes [11] Yes [11] Yes [11] Yes [13] Yes [13] Yes [14] No
p.R255X p.R255X p.Q762X p.D286X
Arg-Term Arg-Term Gln-Term Asp-Term
Tumor Biol.
Database searches further indicated that the p.D286X was novel and closely associated with RB, rather than being widely observed single-nucleotide polymorphisms.
The p.G125X (c.373G>T) mutation is a novel single base change that had been reported [12], and a deletion at this site has also been reported by others [15], which
Tumor Biol. Fig. 2 Pedigree, genomic analysis, and molecular modeling of the novel mutations. Affected individuals with the p.Q344L (a) and p.D286X (b) mutations are indicated by filled symbols. c, d The DNA sequence results of the p.Q344L and p.D286X mutation. The sequence positions of the mutations are indicated with black arrows. e–h, i–l Two molecular structures of p.Q344L and p.D286X and the corresponding wild-type structures. The molecular modeling of p.Q344L is shown in e and f and includes the amino acid substitution and premature termination; the wild-type region around p.Q344L is shown in g and h, and the mutated regions are marked with white arrows. The molecular modeling of p.D286X is shown in i and j, and the wildtype protein is shown in k and l. The white arrows indicate the molecular structure of the wildtype RB1 that is lacking in the mutants
provides evidence that codon 373 is a recurrent mutation site in RB1. Molecular modeling of the pRB RB protein consists of several different domains including a 379 residue N-terminal domain colored in blue (Fig. 1a), a 406 residue central domain, and the pocket domain, which includes an inter-domain linker between the two independently folded domains (RbIDL) and the large loop within the pocket domain (RbPL). The pocket domain extends from residue 379 to 792 and comprises the A and B cyclin-like domains, shown in yellow (Fig. 1a); this domain plays an important role in RB1 function. The summary of the schematic diagram of the mutations (p.Q762X, p.R255X, p.G125X) in RB protein is shown in Fig. 1a, which shows both the sequence and molecular structure of the mutant proteins. Computational analysis of the mutated proteins was performed using a three-
dimensional structural model. Figure 1b represents the sequence of the p.G125X mutant, whereas the spatial structure of the mutated portion of the protein is shown, with two special conformations, in Fig. 1e, f; the corresponding wildtype portions of the protein are shown in Fig. 1g, h. Figure 1c shows the sequence of the p.R255X mutant, and the spatial conformations are shown in Fig. 1i, j; the corresponding wildtype portions of the protein are labeled in Fig. 1k, l. The sequence of the p.Q762X mutant is shown in Fig. 1d, and the mutant and wild-type spatial conformations are in Fig. 1m–p, respectively. The novel mutation p.Q344L led to an amino acid change, glutamine to leucine, and a truncated protein due to the introduction of a premature stop codon (Fig. 2e–f). The p.D286X (c.856-857insert2nt) mutation was also novel and was found in exon 8 of a bilateral RB patient with no family history of RB, which led to the introduction of a premature stop codon, resulting in a protein of only 286 amino acids (Fig. 2i, j). The white arrows indicate the parts of
Tumor Biol.
the wild-type protein that are deleted in the mutants. All of these nonsense mutations resulted in the absence of the RB protein pocket structure.
Subcellular location and the expression of RB and pRB The mutant plasmids p.Q344L and p.D286X (Fig. 3b, d) were transfected into the Saos-2 and 293T cell lines. RB protein expression was detected in Saos-2 cells transfected with wildtype RB1 but not in cells transfected with the mutant plasmids p.Q344L and p.D286X (Fig. 3e). To further investigate the effects of the mutations (p.Q344L and p.D286X) on the subcellular location of RB protein, we performed localization studies in 293T cells. As shown in Fig. 3, wild-type RB protein was localized exclusively in the nucleus in a diffuse manner. In contrast, the cells transfected with the mutant constructs (p.Q344L and p.D286X) did not display a wildtype distribution.
Fig. 3 Expression and location of wild-type and mutant RB1 in transfected Saos-2 and 293T cells. a, c Wild-type sequences of RB1 corresponding to the p.Q344L and p.D286X mutations. b, d Partial plasmid sequence of the constructed plasmids encoding the p.Q344L and p.D286X mutant proteins, respectively. e Expression of RB protein using Western blot after transfection with the wild-type and mutant plasmids, p.Q340L and p.D286X in Saos-2 cells. f Subcellular locations of the wild-type and mutant proteins determined by immunofluorescence after transfected in 293T cells. The left panel corresponds to the most representative subcellular localization of RB as a fusion protein with EGFP-tagged. The middle panel corresponds to nuclear staining with DAPI. The right panel is a merged image of the previous two images. Nuclear fluorescence was obtained with the wild-type protein RB1-EGFP. After transfection of RB-D286XEGFP and RB-Q340L-EGFP, nuclear aggregation and cytoplasmic mislocalization were observed in 293T cells but not in cells transfected with wild-type RB-EGFP
Cell cycle distribution and apoptotic function of the mutants Cell cycle distribution was examined through flow cytometry after transfection with the plasmids. After 48 h in culture, 64 % of the cells transfected with the wild-type RB1 plasmid were blocked in the G1 phase, compared with the control cultures that showed 52 % of cells in the G1 phase (p
RESEARCH ARTICLE
Novel mutations in the RB1 gene from Chinese families with a history of retinoblastoma Leilei Zhang & Renbing Jia & Junyang Zhao & Jiayan Fan & YiXiong Zhou & Bing Han & Xin Song & Li Wu & He Zhang & Huaidong Song & Shengfang Ge & Xianqun Fan
Received: 12 August 2014 / Accepted: 13 November 2014 # International Society of Oncology and BioMarkers (ISOBM) 2014
Abstract Retinoblastoma is an aggressive eye cancer that develops during infancy and is divided into two clinical types, sporadic and heritable. RB1 has been identified as the only pathological gene responsible for heritable retinoblastoma. Here, we identified 11 RB1 germline mutations in the Han pedigrees of 17 bilateral retinoblastoma patients from China. Four mutations were nonsense mutations, five were splice site mutations, and two resulted in a frame shift due to an insertion or a deletion. Three of the mutations had not been previously reported, and the p.Q344L mutation occurred in two generations of retinoblastoma patients. We investigated phenotypic– genotypic relationships for the novel mutations and showed that these mutations affected the expression, location, and function of the retinoblastoma protein. Abnormal protein Leilei Zhang, Renbing Jia, and Junyang Zhao contributed equally to this article. L. Zhang : R. Jia : J. Fan : Y. Zhou : X. Song : H. Zhang : S. Ge (*) : X. Fan (*) Department of Ophthalmology, Shanghai Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine, No. 639 Zhi Zao Ju Road, Shanghai 200011, People’s Republic of China e-mail: [email protected] e-mail: [email protected] J. Zhao Beijing Tongren Eye Centre, Beijing Ophthalmology & Visual Sciences Key Lab, Tongren Hospital, Capital Medical University, Beijing Economic-Technological Development Area, Beijing 100176, People’s Republic of China B. Han : H. Song Department of Endocrinology, Shanghai Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine, No. 639 Zhi Zao Ju Road, Shanghai 200011, People’s Republic of China L. Wu Department of Ophthalmology, People’s Hospital, Wu Han University, No. 238 Jie Fang Road, Wu Han 430060, People’s Republic of China
localization was observed after transfection of the mutant genes. In addition, changes in the cell cycle distribution and apoptosis rates were observed when the Saos-2 cell line was transfected with plasmids encoding the mutant RB1 genes. Our findings expand the spectrum of known RB1 mutations and will benefit the investigation of RB1 mutation hotspots. Genetic counseling can be offered to families with heritable RB1 mutations. Keywords Retinoblastoma . RB1 . Mutation . Genetic counseling
Introduction Retinoblastoma [MIM 180200; RB] [1] is the most common intraocular malignancy in children, with an incidence ranging from 1 in 15,000 to 1 in 20,000 live births [2]. There is no racial or gender predisposition for the development of RB, which is currently split into two clinical types: sporadic and heritable. The average age at the time of diagnosis is 23 months, with unilateral cases being diagnosed at approximately 27 months and bilateral cases at 15 months [3]. All bilateral RB cases are heritable, but only a small proportion of unilateral cases can be passed on to future generations [4]. Hereditary RB, which accounts for 40 % of cases, is transmitted as an autosomal dominant trait that presents as bilateral disease, due to high expressivity (90 %). In 1971, Knudson proposed the “two-hit hypothesis,” which was confirmed in 1986 by the discovery of the RB1 [NM_000321] gene in region 14 of chromosome 13 by Dryja et al. [5]. One allele is mutated in the germline and predisposes the child to retinal tumors, while the second allele is mutated at the cellular level and initiates tumor progression. However, in the nonhereditary disease, both alleles are mutated at the cellular level. RB metastases are rare in the developed world, where early
Tumor Biol.
diagnosis can result in a cure for most children. RB confined to the eye can be cured through simple enucleation. However, curing RB that has metastasized out of the eye is rare [6]. The RB1 gene, located on chromosome 13q14, was the first tumor suppressor gene to be identified. The RB1 gene contains 27 exons distributed over 200 kb of DNA [7] and encodes the retinoblastoma protein [RB protein; NP_000312]. Knowledge of RB1 gene mutations is important for genetic counseling and the characterization of phenotypic–genotypic relationships. To date, more than 900 RB1 germline mutations have been published, and nearly 40 % of RB1 mutations are recurrent [8] and frequently cause gene inactivation. Despite missense mutations being the main inactivating events in most genetic diseases, deletions and nonsense mutations are more often responsible for RB1 inactivation. In 2005, a review indicated that the nonsense mutation rate had reached 42 % [8]. Approximately 60 % of RB1 nonintermediary germline mutations can result in sporadic cases of unilateral disease, with no family history of RB. These RB1 mutations arise locally within the developing retina [9]. However, once an RB1 mutation is present in the germline, it can result in the hereditary transmission of this disease. Individuals with hereditary RB have a 50 % chance of passing these mutations on to their offspring. An RB1 mutation also results in an increased risk of secondary malignancies in the lung, bladder, bone, soft tissues, skin, and brain throughout the patient’s life, particularly when the children are treated with radiation [1]. These secondary, nonocular malignancies are more lethal than RB. In this study, we performed genetic screening of Chinese RB patients and their family members for heritable RB1 mutations. Direct sequencing was used to analyze the RB1 gene and explore potential mutation hotspots. We also investigated the phenotypic–genotypic relationships of two novel mutations in human Saos-2 cells. To the best of our knowledge, this is the first description of these novel mutations.
Materials and methods Subjects and DNA extraction All DNA samples were obtained from the Department of Shanghai Ninth Hospital, and genetic counseling was provided to all participants. Informed consent was obtained from each family member prior to participation in the study, under the authority of the Institutional Ethics Committee of Shanghai Ninth Hospital. Fresh venous blood samples were collected from the patients and their family members and 100 individuals with neither a personal nor family history of RB. The 100 nonRB samples were included as controls to exclude the possibility that the mutations were common polymorphisms. All control samples were from Han Chinese people having the same genetic background. Whole blood was
collected in EDTA tubes. Genomic DNA was extracted from peripheral blood leukocytes (5 ml) using the Automatic Nucleic Acid Isolation System (QuickGene-610L, Fujifilm Life Science, Tokyo, Japan). PCR and sequencing Screening for RB1 mutations was performed through direct polymerase chain reaction (PCR) sequencing of the 27 coding exons and their flanking intron regions. The primer sets and PCR annealing temperatures are described in Table 3. PCR reactions were performed in a thermal cycler in a total volume of 20 μl containing 100 ng of genomic DNA, 5 pmol of each primer, and 7.5 μl of 2× Master Mix (AmpliTaq Gold 360 Master Mix; Applied Biosystems, Foster City, CA, USA). The PCR cycling conditions included an initial denaturation at 95 °C for 5 min followed by 35 cycles of denaturation at 94 °C for 1 min, annealing at the specific temperature for 1 min, 72 °C for 1 min, followed by a final extension step of 72 °C for 7 min. The presence of a specific PCR product was confirmed using 1.5 % agarose gel electrophoresis. Reactions that displayed single bands, without nonspecific amplification, were chosen for sequencing. The mutations were confirmed by sequencing in both the forward and reverse directions on an ABI 3730XL DNA sequencer (Applied Biosystems PerkinElmer). In some cases, a second amplification was required to confirm the mutation. Molecular modeling RB1 mutations can lead to an amino acid substitution in RB protein. To predict the possible impact of a substitution on the structure and function of RB protein, a three-dimensional computer model was used to analyze the structural positioning of the mutation sites. The crystal structure of the DNAbinding domain of RB protein was obtained from the Protein Data Bank (PDB) (code, 4ELJ, chain A). The DNAbinding domain of RB protein was modeled on the basis of homology using SWISS-MODEL, as previously described [10]. Construction of the expression vectors Professor Zhao (Southern Medical University, Key Laboratory for Proteomics of in Guangdong Province) provided a plasmid that contained the wild-type RB1 gene. The wild-type RB1 gene was attached to an enhanced green fluorescent protein (EGFP) tag, and the wild-type RB1 and wild-type RB1-EGFP plasmids were used as template to construct the mutant plasmids. The mutations without the EGFP tag were used for fluorescein isothiocyanate (FITC)/Annexin V apoptosis staining cell cycle distribution analysis, while the mutants with the
Tumor Biol.
EGFP tag were used for subcellular location analysis. The mutant expression vectors were generated using site-specific mutagenesis, with the wild-type plasmids as templates. The following primers were used for p.D286X: sense 5′-CATGAA TGTAATATATAGATGAGGTG-3′ and antisense 5′-TATATA TTACATTCATGTTCTTTACAGAG-3′. The following primers for p.Q344L were used: sense 5′-ATGATAAAACTC TTCTGATTCTATAGACAG-3′ and antisense 5′-GAAGAG TTTTATCATGATCCAAAAA-3′. The reactions were performed using a touchdown PCR protocol with an initial denaturation of 95 °C for 5 min followed by 16 cycles of denaturation at 94 °C for 50 s, annealing at 64 °C for 1 min, and elongation at 68 °C for 11 min. After each cycle, the annealing temperature was decreased by 0.5 °C. An additional 16 cycles of denaturation at 94 °C for 50 s, annealing at 56 °C for 1 min, and extension at 68 °C for 11 min were performed, followed by a final extension step at 68 °C for 11 min. The KOD-Plus-Neo enzyme (TOYOBO, Osaka, Japan) was used to perform the touchdown PCR reactions. After completion of the PCR reaction, the wild-type plasmid was removed with the Dpn1 enzyme (Sangon, Shanghai, China) which only targets methylated sites within the plasmid that are not present in the PCR products. The mutated plasmids were isolated and purified using anion exchange columns (TIANGEN BIOTECH, Beijing, China). All constructs were sequenced and confirmed without single mutations at other sites. Cell culture and transfection The Saos-2 cell line was kindly provided by Professor Hao (Shanghai, China). The 293T cell line was purchased from the American Type Culture Collection (Manassas, VA, USA). Both Saos-2 and 293T cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, CA, USA), supplemented with 10 % fetal bovine serum (FBS; Gibco– Invitrogen, Grand Island, NY, USA) in a 5 % CO2 atmosphere. Transfection was performed using Lipofectamine 2000 (Invitrogen, CA, USA), according to the manufacturer’s instructions. A day before transfection, 2×105 cells were plated in 2 ml of growth medium, without antibiotics, to ensure that the cells were 50–80 % confluent at the time of transfection. Transfection was performed the following day; afterwards, the serum-containing culture medium was changed to serum-free culture medium. Four hours after transfection, the serum-free culture medium was replaced to serumsupplemented culture medium. Cell cycle analysis Wild-type and mutant plasmids were transfected into Saos-2 cells, which were harvested 48 h after transfection, along with control cells that had not undergone transfection. The cells were washed twice with cold phosphate-buffered saline
(PBS), fixed in 70 % ethanol, and stored at 4 °C overnight. On the next day, they were then washed twice with cold PBS and incubated with propidium iodide/ribonuclease staining solution (Becton Dickinson, NJ, USA) for 15 min at room temperature, following the manufacturer’s instructions. Cell cycle distribution was detected and analyzed using the FACScan instrument and CellQuest program (Becton Dickinson, NJ, USA). Apoptosis analysis Forty-eight hours after transfection, the cells were harvested and washed in cold PBS. Apoptosis was determined through flow cytometry using the FITC/Annexin V Apoptosis Detection Kit (Becton Dickinson, NJ, USA), according to the manufacturer’s instructions. Samples were examined by the FACScan instrument (Becton Dickinson, NJ, USA), and data were analyzed using the CellQuest programs (Becton Dickinson, NJ, USA). Immunofluorescence 293T cells transfected with plasmids encoding wild-type RB1 or the p.D286X and p.Q344L RB1 mutants were seeded onto glass coverslips (VWR, West Chester, PA, USA) in 12-well plates and cultured overnight. Forty-eight hours after transfection, cells were fixed in 4 % (w/v) paraformaldehyde (SigmaAldrich, St. Louis, MO, USA) for 15 min at room temperature. The coverslips were washed again, and the nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI; Vector Laboratories, Burlingame, CA, USA) for 2 min at room temperature. The control samples were processed using the same protocol, but the primary antibody was omitted. Immunoreactive cells were visualized using a fluorescent microscope (Olympus BX51, Japan). Western blot (immunoblot) analysis The protein extracted from cells and supernatants was collected by centrifugation at 12,000×g at 4 °C for 30 min. The total protein concentration was determined using the BCA protein assay kit (Pierce, Rockford, IL, USA). Equal amounts of total cellular extracts (20 μg) were separated by SDS-PAGE and blotted onto PVDF membranes (Millipore, USA). After the addition of blocking solution (5 % nonfat dry milk/0.1 % Tween-20/PBS), the membranes were incubated overnight with the recommended dilution of the following primary antibodies: anti-RB1 (1:1,000, Epitomics, USA); anti-activatedcaspase-3 (1:1,000, caspase-3 p17, Bioworld Technology, USA); and anti-ß-actin (1:10,000, Invitrogen, USA). After incubation with the primary antibodies at 4 °C overnight, the membranes were washed three times with TBS containing 0.1 % Tween-20 (TBST) and incubated with a secondary
Tumor Biol.
antibody (Invitrogen, USA) conjugated to a fluorescent tag for 1 h. The membranes were then washed three times with TBST. The bands were detected using the Odyssey infrared imaging system (Odyssey; LI-COR, Lincoln, NE, USA). Cell viability Cell viability was quantified using cell counting kit-8 (CCK8, Dojindo, Kumamoto, Japan) according to the manufacturer’s instruction. In brief, Saos-2 cells were plated to 96-well plates at a concentration of 2,000 cells/well with five duplicates. After overnight, the cells were transfected with wild-type RB1 and mutants. Four hours later, the medium was changed to DMEM with 10 % FBS and then treated for 24, 48, and 72 h. Absorbance was measured after a further 3-h incubation at 37 °C with an addition of cell counting kit-8 (10 μg/ml). The absorbance was measured on a spectrophotometer microplate reader at a wavelength of 450 nm.
Results Genetic analysis DNA from the RB patients was analyzed using reverse transcription PCR and was sequenced to obtain the complete coding sequence of the RB1 gene. Seventeen RB patients were analyzed, and 11 mutations were detected. Two mutations were novel with one recurred twice and nine had been reported previously (Table 1). Five mutations were single base substitutions (p.Q762X, p.D286X, p.R255X (observed twice), and p.G125X) that led to nonsense amino acid changes (Fig. 1a). The previously reported splice site mutations
Table 1
Fig. 1 A summary of the RB1 gene mutations in RB patients. a Schematics show the human wild-type and mutant versions of RB1. The N-terminal domain is shown in blue. The yellow portion of the diagram indicates the pocket-like structures of RB protein, which extends from residue 379 to 792 and plays an important role in RB1 function. The mutants are shown below including p.Q762X, p.Q344L, p.D286X, p.R255X, and p.G125X, with the C-terminal domain removed. With the exception of p.Q762X, the pocket-like domain of other mutants is removed. b–d The sequences of the p.G125X, p.R255X, and p.Q762X mutations. e–h, i–l, and m–p The two structure types of the mutants and the corresponding wild-type sequences. e, g, i, k, m, and o The space filling conformations. f, h, j, l, n, and p The ribbon conformation, e, f protein structure of the p.G125X mutant. i, g protein structures of p.R255X, and m, n molecular modeling of p.Q762X, all of which lead to the premature termination of the protein. g, h, k, l, o, and p Corresponding wild-type protein structures of p.G125X, p.R255X and p.Q762X, respectively, which are shown without deletion of the partial protein structure (indicated by white arrows)
(IVS22 as −2 A-G) were observed five times in different patients in this study. One novel mutation led to the deletion of four base pairs, AGAC (c.1031_1034del4nt, p.Q344L), in the open reading frame of a male patient. The family history of this patient was positive for RB, and the same mutation was present in the boy’s father, which is an inherited mutations. The pedigree of this family is shown in Fig. 2a. This variant was not observed in 100 control individuals or the patient’s mother. This result indicated that p.Q344L may play an important role in the initiation and progression of RB in this family. In addition, several databases, including NCBI, dbSNP, and UCSC, were searched to ensure that this substitution was not a single-nucleotide polymorphism. Our findings indicated that this was indeed a novel mutation that was closely associated with RB in this Chinese family. The p.D286X (c.856-857insert2nt) mutation was also novel and was found in exon 8 of a bilateral RB patient with no family history of RB (Fig. 2b), which inserted two nucleotides (TA).
RB1 germline mutations identified in Chinese patients with retinoblastoma
No.
RB phenotype
Type of mutation
cDNA
Protein
Exon location
Functional consequences
Reported
1a-10 1a-23
Bilateral Bilateral
Deletion Splicing
c.1031_1034del4nt IVS22 as −2 A-G
p.Q344 L
10 23
Gln-Leu
No Yes [11]
2a-3 2a-23 3a-23 5a-23 6a-23 3a-8 12a-8 4a-22 5a-8
Bilateral Bilateral Bilateral Bilateral Bilateral Bilateral Bilateral Bilateral Bilateral
Nonsense Splicing Splicing Splicing Splicing Substitution Substitution Nonsense Insertion
c.373 G>T IVS22 as −2 A-G IVS22 as −2 A-G IVS22 as −2 A-G IVS22 as −2 A-G c.763 C>T c.763 C>T c.2284 C>T c.856-857insert2nt
p.G125X
3 23 23 23 23 8 8 22 8
Glu-Term
Yes [12] Yes [11] Yes [11] Yes [11] Yes [11] Yes [13] Yes [13] Yes [14] No
p.R255X p.R255X p.Q762X p.D286X
Arg-Term Arg-Term Gln-Term Asp-Term
Tumor Biol.
Database searches further indicated that the p.D286X was novel and closely associated with RB, rather than being widely observed single-nucleotide polymorphisms.
The p.G125X (c.373G>T) mutation is a novel single base change that had been reported [12], and a deletion at this site has also been reported by others [15], which
Tumor Biol. Fig. 2 Pedigree, genomic analysis, and molecular modeling of the novel mutations. Affected individuals with the p.Q344L (a) and p.D286X (b) mutations are indicated by filled symbols. c, d The DNA sequence results of the p.Q344L and p.D286X mutation. The sequence positions of the mutations are indicated with black arrows. e–h, i–l Two molecular structures of p.Q344L and p.D286X and the corresponding wild-type structures. The molecular modeling of p.Q344L is shown in e and f and includes the amino acid substitution and premature termination; the wild-type region around p.Q344L is shown in g and h, and the mutated regions are marked with white arrows. The molecular modeling of p.D286X is shown in i and j, and the wildtype protein is shown in k and l. The white arrows indicate the molecular structure of the wildtype RB1 that is lacking in the mutants
provides evidence that codon 373 is a recurrent mutation site in RB1. Molecular modeling of the pRB RB protein consists of several different domains including a 379 residue N-terminal domain colored in blue (Fig. 1a), a 406 residue central domain, and the pocket domain, which includes an inter-domain linker between the two independently folded domains (RbIDL) and the large loop within the pocket domain (RbPL). The pocket domain extends from residue 379 to 792 and comprises the A and B cyclin-like domains, shown in yellow (Fig. 1a); this domain plays an important role in RB1 function. The summary of the schematic diagram of the mutations (p.Q762X, p.R255X, p.G125X) in RB protein is shown in Fig. 1a, which shows both the sequence and molecular structure of the mutant proteins. Computational analysis of the mutated proteins was performed using a three-
dimensional structural model. Figure 1b represents the sequence of the p.G125X mutant, whereas the spatial structure of the mutated portion of the protein is shown, with two special conformations, in Fig. 1e, f; the corresponding wildtype portions of the protein are shown in Fig. 1g, h. Figure 1c shows the sequence of the p.R255X mutant, and the spatial conformations are shown in Fig. 1i, j; the corresponding wildtype portions of the protein are labeled in Fig. 1k, l. The sequence of the p.Q762X mutant is shown in Fig. 1d, and the mutant and wild-type spatial conformations are in Fig. 1m–p, respectively. The novel mutation p.Q344L led to an amino acid change, glutamine to leucine, and a truncated protein due to the introduction of a premature stop codon (Fig. 2e–f). The p.D286X (c.856-857insert2nt) mutation was also novel and was found in exon 8 of a bilateral RB patient with no family history of RB, which led to the introduction of a premature stop codon, resulting in a protein of only 286 amino acids (Fig. 2i, j). The white arrows indicate the parts of
Tumor Biol.
the wild-type protein that are deleted in the mutants. All of these nonsense mutations resulted in the absence of the RB protein pocket structure.
Subcellular location and the expression of RB and pRB The mutant plasmids p.Q344L and p.D286X (Fig. 3b, d) were transfected into the Saos-2 and 293T cell lines. RB protein expression was detected in Saos-2 cells transfected with wildtype RB1 but not in cells transfected with the mutant plasmids p.Q344L and p.D286X (Fig. 3e). To further investigate the effects of the mutations (p.Q344L and p.D286X) on the subcellular location of RB protein, we performed localization studies in 293T cells. As shown in Fig. 3, wild-type RB protein was localized exclusively in the nucleus in a diffuse manner. In contrast, the cells transfected with the mutant constructs (p.Q344L and p.D286X) did not display a wildtype distribution.
Fig. 3 Expression and location of wild-type and mutant RB1 in transfected Saos-2 and 293T cells. a, c Wild-type sequences of RB1 corresponding to the p.Q344L and p.D286X mutations. b, d Partial plasmid sequence of the constructed plasmids encoding the p.Q344L and p.D286X mutant proteins, respectively. e Expression of RB protein using Western blot after transfection with the wild-type and mutant plasmids, p.Q340L and p.D286X in Saos-2 cells. f Subcellular locations of the wild-type and mutant proteins determined by immunofluorescence after transfected in 293T cells. The left panel corresponds to the most representative subcellular localization of RB as a fusion protein with EGFP-tagged. The middle panel corresponds to nuclear staining with DAPI. The right panel is a merged image of the previous two images. Nuclear fluorescence was obtained with the wild-type protein RB1-EGFP. After transfection of RB-D286XEGFP and RB-Q340L-EGFP, nuclear aggregation and cytoplasmic mislocalization were observed in 293T cells but not in cells transfected with wild-type RB-EGFP
Cell cycle distribution and apoptotic function of the mutants Cell cycle distribution was examined through flow cytometry after transfection with the plasmids. After 48 h in culture, 64 % of the cells transfected with the wild-type RB1 plasmid were blocked in the G1 phase, compared with the control cultures that showed 52 % of cells in the G1 phase (p
