Novel point mutation and intronic mutations of RB1 gene in retinoblastoma patients in Indonesia

Batari Todja Umar,1 Ulfah Rimayanti,1,2 Halimah Pagarra,1 Budu,1,3 Nasrum Massi,4 Habibah Setyawati Muhiddin1

Background

Retinoblastoma (RB) is an inherited disorder caused by the RB1 gene mutation in retinal cells or germline mutation. Identifying the specific mutation is crucial for prognosis, inheritance risk assessment, and treatment planning. This study aimed to identify the germline mutation in the RB1 gene in patients with RB and their parents from the eastern part of Indonesia.

 

Methods

This observational analytic study recruited patients with RB and their parents between 2016 and 2018 at Dr. Wahidin Sudirohusodo Hospital, Makassar, Indonesia. The normal control subjects were children from the outpatient clinic at the Department of Ophthalmology, Universitas Hasanuddin Hospital. Ophthalmic examinations and peripheral blood tests were performed in RB patients, their parents, and control subjects. Genomic DNA was isolated from blood leukocytes and amplified using conventional PCR. Hotspot exons 8, 10, 14, 17, and 22 were screened for mutations using the Sanger method.

 

Results

There were 21 patients with RB (16 unilateral and 5 bilateral) and 14 normal subjects. Of the 184 variations detected in RB patients, 164 were also found in normal subjects. 19 intronic mutations in introns 10, 16, 17, and 21, and 1 novel missense mutation in exon 17 were identified. Parental testing revealed 8 substitutions in exon 17 and 5 intronic mutations in introns 16 and 17 of the parents. None of the variations in exons were passed to their children.

 

Conclusions

This study found a novel missense mutation in exon 17 of the RB1 gene.

 

Keywords

germline mutation, RB1 gene, retinoblastoma

Retinoblastoma (RB) is the most common eye neoplasm in childhood,1 with approximately 50% of cases being heritable due to a mutation of the RB1 gene that predisposes the development of retinal tumors. The RB1 gene mutation occurs in retinal cells (somatic mutation) or gametes (germline mutation) in 98% of RB cases.2 This gene comprises 27 exons spanning 178,143 kb genomic DNA at chromosome 13q143,4 and encodes RB protein (pRB), which is a nuclear phosphoprotein and crucial for regulating cell cycle progression.4,5 More than 1,900 mutations in the RB1 gene have been reported globally,6 and identifying these mutations in RB patients is crucial for developing mutational analysis procedures and understanding the molecular mechanism underlying disease penetrance and expressivity.

Familial RB mutations exhibit variable expressivity and phenotypic variability.6–10 The RB1 mutations associated with low penetrance include promoter mutations, missense mutations, and in-frame deletions/insertions.11,12 These mutations are autosomal dominant; thus, children whose parents have the RB1 gene mutation are at a higher risk of developing RB and other cancers such as osteosarcoma, small cell lung carcinoma, bladder tumor, and breast carcinoma.6 Identifying mutation type in patients with RB and their parents is essential for assessing short-term prognosis (intraocular tumor in the same eye or fellow eye), long-term prognosis (the risk of non-ocular malignancies), inheritance risk, and treatment strategy.13,14 Mutational screening of the RB1 gene can contribute to clinical management and provide accurate genetic counseling to patients and their families. Despite the importance of RB1 gene mutational analysis, there is a lack of data about germline RB1 mutations in RB patients in Indonesia. This study aimed to identify the RB1 gene mutations in patients with RB in the eastern part of Indonesia.

 

 

Subjects

All subjects were recruited between 2016 and 2018 at Dr. Wahidin Sudirohusodo Hospital, Makassar, Indonesia, which included patients diagnosed with RB and their parents. The diagnosis of RB was established through standard ophthalmological and histological examinations. Patients with a history of radiotherapy or other malignancies were excluded from the study. The control group was composed of children at the outpatient clinic at the Department of Ophthalmology, Universitas Hasanuddin Hospital, who had no abnormal ophthalmological findings except for mild refractive error and had neither history nor family history of neoplasm.

 

Sample collection and DNA extraction

A total of 5 ml peripheral blood samples were collected from patients and their parents into standard ethylenediaminetetraacetic acid (EDTA) tubes, and the DNA sample was stored at −20°C. The available parental samples were examined for the RB1 gene mutation at the same nucleotide position as their child who had a germline mutation. The blood samples were analyzed at Hasanuddin University Medical Research Center laboratory. DNA was isolated from peripheral blood leukocytes using gSYNC DNA Extraction Kit (Geneaid Biotech Ltd., Taiwan) according to the manufacturer’s protocol.15

 

Sample preparation

Transfer up to 200 μl of whole blood to a 1.5 ml microcentrifuge tube. Adjust the volume to 200 μl with phosphate buffered saline. Add 20 μl of proteinase K then mix by pipetting. Incubate at 60°C for 5 min.

 

Cell lysis

Add 200 μl of gel sample buffer, then mix by shaking vigorously. Incubate at 60°C for 5 min, add proteinase, then incubate again for 5 min.

 

DNA binding

Add 200 μl of absolute ethanol to the sample lysate and mix immediately by shaking vigorously for 10 sec. Transfer all of the mixture to the GS Column. Centrifuge at 14–16,000 × g for 1 min. Transfer the GS Column to a new 2 ml collection tube.

 

Wash

Add 400 μl of W1 buffer to the GS Column. Centrifuge at 14–16,000 × g for 30 sec. Add 600 μl of wash buffer (make sure absolute ethanol was added) to the GS Column. Centrifuge at 14–16,000 × g for 30 sec. Centrifuge for 3 min at 14–16,000 × g.

 

Elution

Transfer the dried GS Column to a 1.5 ml microcentrifuge tube. Add 100 μl of pre-heated elution buffer, Tris-EDTA buffer. Let stand for at least 3 min. Centrifuge at 14–16,000 × g for 30 sec. DNA quality was evaluated with 2% agarose gel electrophoresis at 100 V (Bio-Rad, USA); the DNA quantity was not calculated.

 

Polymerase chain reaction (PCR) and sequencing

Screening of germline mutations in the RB1 gene was performed by direct Sanger sequencing for exons 8, 10, 14, 17, and 22. The primer sequences followed the previously published primer sequences for the RB1 gene, as presented in Table 1.3 The DNA was amplified in a PCR DNA thermal cycler (Bio-Rad, USA) in a total volume of 50 μl. The PCR component was KAPPA2G fast enzyme, forward and reverse RB1 gene primer (Table 1), nuclease-free water, and DNA template. PCR cycling was initiated with the denaturation step at 95°C for 15 min, then 94°C for 1 min, annealing at 55°C for 30 sec, extension at 72°C for 1.5 min as much as 45 cycles, continued with the last extension at 72°C for 10 min and 12°C for 30 min for storage. The PCR product was sequenced to detect mutation in the RB1 gene, then analyzed using BioEdit Sequence Alignment Editor version 7.0.5.1. software (Tom Hall; North Carolina State University, USA). The results were then compared with the data in Gene Bank of National Center for Biotechnology Information GRCh37 database using basic local alignment search tool. Additional information about mutations in the RB1 gene was confirmed from the RB1 variation database RB1-lsdb and the Human Gene Mutation Database.

 

 

 

Statistical analysis

The demographic data between normal subjects and patients with RB were compared using the Mann–Whitney U test. A p-value of <0.05 was considered statistically significant. The statistical analyses were performed using the JMP software version 13.0 (SAS Institute Inc., USA).

 

Ethical considerations

The procedures adhered to the tenets of the Declaration of Helsinki and were approved by the Ethics Committee of the Faculty of Medicine, Universitas Hasanuddin (No. 555/UN4.6.4.5.31/PP36/2019). Written and signed informed consent from the parents was obtained before the examinations.

 

 

A total of 21 patients with RB and 14 normal subjects were included in this study. RB patients were mostly diagnosed at a later stage, with 81% of patients exhibiting tumor extension. Additionally, 14% of patients were classified as Group V according to the Reese-Ellsworth classification, and 5% had a regressed type of RB. Of the patients, 76% had unilateral RB, and 24% had bilateral RB. Table 2 presents the demographic data for these groups.

 

 

Ten patients with unilateral RB and one with bilateral RB had the RB1 gene variations. The mean paternal age was 36 years old, and the mean maternal age was 32 years old. Ten out of 11 fathers had a smoking habit, whereas none of the mothers smoked. Furthermore, the parents had no history of radiation exposure or a family history of RB.

In this study, 184 variations were discovered in RB patients, with 164 of which were also found in normal subjects. Nineteen intronic mutations in introns 10, 16, 17, and 21, and one missense mutation in exon 17 were identified; these mutations were not found in normal subjects. One patient (5%) (R33) had a germline mutation among the 21 patients. The heterozygous mutation identified in exon 17 of R33 is presented in Figure 1, which caused an amino acid change from tryptophan to glycine. No family members of R33 had a history of RB. The mutation list of the RB1 gene in RB patients is shown in Table 3.

 

 

 

 

The analysis of the parental origin of the detected mutations revealed the RB1 gene variations in seven out of 21 parents whose children tested positive for the RB1 gene mutations. The variations included eight substitutions in exon 17 and five intronic mutations in introns 16 and 17, as shown in Table 3. However, none of the variations in exons were passed to their children who had RB.

 

 

This study conducted a comprehensive screening of germline mutations of the RB1 gene in Indonesian patients with RB and found a 5% germline RB mutation rate. The prevalence of the RB1 gene germline mutation varies worldwide, ranging from 37% in Italy to 51% in India among patients with RB. Germline mutation occurs in 51.06, 37.14, and 42.4% of patients with RB in India, Italy, and Singapore, respectively.8,9,16 Moreover, the rate is higher in Vietnam (73.53%).6 The prevalence of germline mutation of the RB1 gene in various countries are presented in Table 4.8–10,16–22

 

 

Notably, this study found a novel missense mutation in exon 17 in patients with a unilateral tumor, but no germline mutation was detected in patients with bilateral RB. A study in Singapore found that 71% of patients with bilateral RB and 29% with unilateral RB had a RB1 gene germline mutation.16 Meanwhile, these mutations were detected in 44% of Vietnamese patients with a unilateral tumor and 84% with a bilateral tumor.6

Exon 17 is crucial as it encodes the domains A and B of the pRB pocket, necessary for repressor activity that blocks cell growth by binding to transcriptional factors E2F/DP.4,23 Mutations in this repressor domain may disturb growth suppression and cause loss of cell growth control. The identified de novo missense mutation in exon 17 induced amino acid changes from tryptophan to glycine. Tryptophan is an essential amino acid with amphipathic properties, exhibiting weak hydrophilic and hydrophobic characteristics.24–26 Consequently, it tends to compose the inner core of soluble proteins.27 Meanwhile, glycine is a non-essential hydrophobic amino acid with unique features, including its backbone consisting of only one hydrogen atom, which makes it more flexible than other amino acids.25–28 Due to this characteristic, glycine is often located at the interface where two polypeptides come into contact. In the RB1 gene, even a single amino acid substitution may result in a dysfunctional pRB, compromising its role as a tumor suppressor gene.29 However, additional analysis such as minigene assay or in silico analysis may be necessary to confirm the pathogenicity of a mutation.30,31 This was another limitation of this study; we did not perform additional assay and pathogenicity assessment. This low prevalence of germline mutation might be due to the inability to identify mutational change in all RB1 gene exons, as some “hot-spot” exons (8, 10, 14, 17, and 22) are more commonly affected.16 Another possibility was due to the small sample size.

This study compared sequencing results between the RB patients and normal subjects (control) to reduce the possibility of normal gene variants or polymorphism. DNA exon primers were used,3 as well as DNA sequence to read the adjacent intronic parts. Of the 20 mutations discovered, 90% were intronic mutations, with deep intronic mutations accounting for 66.67%. Although only a few bases deep in introns are known to be effective targets for oncogenic mutations,32 point mutations deep within introns (>100 bp from a splice junction) may alter normal splicing, transcription regulatory motifs, and non-coding RNA genes.33–35 Some promoters also collaborate with regulatory sequences within the intron.36

In most cases, RB was unilateral and sporadic, and the parents appeared genetically normal. In some families, carriers had no tumor (reduced penetrance) or only unilateral RB or benign retinocytoma (reduced expressivity).11 These variations in the phenotype may be caused by various factors such as immunologic factors, epigenetic mechanisms, delayed mutation, host resistance, and modulatory genes.37–41

Our findings have implications for prognosis and genetic counseling for the patients and their families. We provided genetic counseling to one pregnant patient with unilateral regressed RB (R3), and no germline mutation was detected. However, we advised her about the possibly inheriting the condition on to her offspring, even though the risk was smaller than for patients with a germline mutation. Since most parents were of their active reproductive age, comprehensive genetic counseling about RB is crucial

In conclusion, one novel missense mutation was identified in exon 17 of the RB1 gene, which was not detected in normal subjects. This finding has implications for prognosis, genetic counseling, and risk prediction for affected families. Additional studies are needed to confirm the pathogenicity of the mutation and explore other mutational changes in the entire RB1 gene sequencing.

 

 

 

 

 

 

 

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