Ophthalmic Genetics
ISSN: 1381-6810 (Print) 1744-5094 (Online) Journal homepage: http://www.tandfonline.com/loi/iopg20
Prenatal genetic diagnosis of retinoblastoma and report of RB1 gene mutation from India Parag K. Shah, S. Sripriya, V. Narendran & A. J. Pandian To cite this article: Parag K. Shah, S. Sripriya, V. Narendran & A. J. Pandian (2016): Prenatal genetic diagnosis of retinoblastoma and report of RB1 gene mutation from India, Ophthalmic Genetics, DOI: 10.3109/13816810.2015.1107595 To link to this article: http://dx.doi.org/10.3109/13816810.2015.1107595
Published online: 25 Feb 2016.
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Date: 26 February 2016, At: 21:10
OPHTHALMIC GENETICS http://dx.doi.org/10.3109/13816810.2015.1107595
CASE REPORT
Prenatal genetic diagnosis of retinoblastoma and report of RB1 gene mutation from India Parag K. Shaha, S. Sripriyab, V. Narendrana, and A. J. Pandianb
Downloaded by [University of California, San Diego] at 21:10 26 February 2016
a Department of Pediatric Retina & Ocular Oncology, Aravind Eye Hospital & Postgraduate Institute of Ophthalmology, Coimbatore, Tamilnadu, India; bSankara Nethralaya ONGC Department of Genetics and Molecular Biology, Vision Research Foundation, Chennai, Tamilnadu, India
ABSTRACT
ARTICLE HISTORY
Background: Retinoblastoma is the most common intraocular malignancy of childhood. There is a paucity of genetic testing and prenatal genetic diagnosis from India, which has the highest incidence worldwide. Materials and methods: RB1 gene screening of an 8-month-old female child with bilateral retinoblastoma was accomplished using next generation sequencing. The results were used for prenatal testing in this family. Results: A heterozygous germline mutation (chr13: 48951119delA; c.1281delA) was detected, which resulted in premature termination of a protein product (p.Glu428Argfs*29). Prenatal testing in maternal DNA revealed carrier status of the mother. Further clinical examination in the family members revealed retinocytomas in both eyes of the mother and maternal grandmother. Prenatal genetic testing of the developing fetus showed positivity for the mutation. As the family preferred to continue the pregnancy, serial 3-D ultrasounds were carried out every 2 weeks in the third trimester. Ten days after delivery, small extrafoveal tumors developed in both eyes, which were then treated successfully with transpupillary thermotherapy. Conclusion: We report the significance of genetic testing in the early detection and management of retinoblastoma from India.
Received 6 March 2015 Revised 27 September 2015 Accepted 3 October 2015
Introduction Retinoblastoma is a genetic disease caused by mutation in the RB1 gene. More than 90% of cases are sporadic while less than 10% are familial.1 Approximately 40% of cases have germline mutations, while the remaining 60% have somatic mutations.2 Prenatal genetic diagnosis of retinoblastoma has been reported previously.3,4 There are very few reports from India,5 which has the highest incidence of retinoblastoma in the world.6 We report one of the first cases of prenatal genetic diagnosis from India.
Case report An 8-month-old female child was brought to our hospital with complaints of squinting of eyes since birth. Pedigree analysis revealed a history of an eye tumor, likely retinoblastoma and childhood death of a maternal aunt. The child was able to fix and follow light with both eyes open. She resisted occlusion of the right eye. Anterior segment examination showed mild esotropia along with nystagmus in both eyes. Fundus examination of the right eye under anaesthesia showed a large whitish mass lesion involving the entire macular region with surrounding exudative retinal detachment and diffuse subretinal seeds. The left eye fundus showed total exudative detachment with a large subretinal mass (Figure 1). Ultrasonography of both eyes showed large mass lesions with intralesional calcification. Magnetic resonance
KEYWORDS
India; prenatal; RB1 gene; retinoblastoma
imaging of the brain showed no extraocular spread. A diagnosis of bilateral retinoblastoma (group D in the right eye and group E in the left eye) was made, according to the International Retinoblastoma Classification.7 The child was given six cycles of systemic chemotherapy using carboplatin, vincristine and etoposide, along with three injections of subTenon’s carboplatin to the right eye and transpupillary thermotherapy (TTT) to both eyes. At the end of the chemotherapy cycles, the tumors of both eyes showed type 3 regression (which is partial calcification with cottage cheese appearance of the tumor) while the vitreous seeds were calcified suggestive of type 1 vitreous seeds regression (which is refringent and/or calcified vitreous seeds residues).8 However 10 weeks later, tumor recurrence was observed in both eyes and as parents refused enucleation of the left eye, external beam radiotherapy (EBRT) was given at a dose of 36 Gy to both eyes. Following EBRT, both eyes showed good regression. Fundus examination of the mother showed large peripheral chorioretinal lesions with foci of calcification in both the eyes, suggestive of presumed retinocytomas (Figure 2). Fundus examination of the maternal grandmother of the proband also showed presumed retinocytomas in both eyes (Figure 3). RB1 gene screening was performed by Next Generation Sequencing (NGS) using the MiSeqIllumina NGS platform (Strand Life Science Pvt Ltd, Bangalore, India). Variations were identified by using the AVADIS® NGS software and interpreted using the StrandOmics tool. The observed variation was further validated by direct sequencing
CONTACT Parag K. Shah [email protected] Department of Pediatric Retina & Ocular Oncology, Aravind Eye Hospital & Postgraduate Institute of Ophthalmology, Avinashi Road, Coimbatore, Tamilnadu 641014, India. Color versions of one or more figures in the article can be found online at http://www.tandfonline.com/iopg. © 2016 Taylor & Francis
2
P. K. SHAH ET AL.
Downloaded by [University of California, San Diego] at 21:10 26 February 2016
Figure 1. (A) Retcam picture of the right eye showing a large macular retinoblastoma. (B) Left eye showing total exudative retinal detachment with a subretinal mass.
Figure 2. (A) Fundus picture of the right eye of the mother showing para macular flat lesions with calcification (white arrow) suggestive of retinocytoma. (B) Fundus picture of left eye showing retinocytoma (white arrow) in the temporal periphery.
Figure 3. (A) Fundus picture of the right eye of the grandmother showing a large retinocytoma involving the macula (white arrow). (B) Fundus picture of the left eye showing retinocytoma (white arrow) in the temporal periphery.
and cosegregation analysis was performed in the pedigree members. Next generation sequencing analysis of the RB1 gene in the illumina platform revealed a heterozygous deletion (chr13: 48951119delA c.1281delA) that resulted in the premature truncation of the retinoblastoma protein (lu428Argfs*29). Analysis of the mother’s DNA revealed the same heterozygous mutation and the father was normal (Figure 4). As the family wanted to have another child, after genetic counseling, prenatal diagnosis was offered. The fetal blood sample was collected by amniocentesis and tested for the specific known mutation by direct sequencing. Multi loci short tandem repeats (STR) analysis was performed to rule out maternal contamination. The fetal DNA was positive for the mutation in the heterozygous state. As they wanted to continue the pregnancy, serial 3-D ultrasounds were performed every 2 weeks during the third trimester.9 No eye abnormalities were observed on prenatal ultrasound imaging and child was delivered at full term. Fundus examination 3
days after birth revealed multiple birth-related retinal hemorrhages. No tumors were seen. Repeat examination at 10 days showed three new, small tumors in the right eye and two new tumors in the left eye (Figure 5). All were less than 3 mm in size. As none involved the fovea, immediate TTT was performed. Following this treatment, all the tumors showed a flat scar until the last follow up at 3 months.
Discussion India has the highest reported incidence of retinoblastoma.6 Of the predicted global burden of 8099 cases of retinoblastoma in 2013, India had 1486 cases, followed by China with 1103 cases.6 Although the survival rates in India have greatly increased,2,10 there is a paucity of genetic testing. With increased survival, we now see second and even third generations of children born to survivors. The potential benefits of genetic counseling and prenatal testing for retinoblastoma has
Downloaded by [University of California, San Diego] at 21:10 26 February 2016
OPHTHALMIC GENETICS
3
Figure 4. Cosegregation analysis of the c.1281delA mutation in exon 13 of the RB1 gene. Samples II1, III1 and III2 are positive for the nucleotide variation.
Figure 5. (A & B) Retcam fundus pictures of the right and left eyes at 10 days after birth showing small, round, creamy masses (white arrows) consistent with retinoblastoma.
previously been reported in a handful of cases in India.5With increased availability and awareness of this testing, prenatal genetic diagnosis is being used with increased frequency.2 Indications for prenatal screening include: the presence of bilateral retinoblastoma in either parent, retinocytoma in either parent (as in our case), or a positive family history of retinoblastoma. Methods of prenatal screening are preimplantation genetic diagnosis (PGD),11,12 prenatal genetic testing of the fetus, prenatal ultrasound, and fetal magnetic resonance imaging (MRI).9 PGD is done on 12-day-old embryos after in vitro fertilization. Unaffected embryos are then transferred to the uterus to continue the pregnancy. PGD although sensitive, is technically challenging and limited only to certain medical centers in the world. Both prenatal ultrasound and fetal MRI have limitations, in that they may not detect minimally elevated retinoblastoma tumors. Prenatal ultrasound is started between 16 and 18 weeks gestation, and repeated every 4 weeks until 32 weeks, then every 2 weeks until delivery. Fetal MRIs are performed every 8 weeks starting at 16–18 weeks of gestation.9 Compared to fetal MRI, prenatal
ultrasound is more sensitive for the detection of retinoblastoma tumors.9 If tumors are detected prenatally, then preterm delivery can be considered as an option, so that immediate treatment can be initiated. Delivery before 34 weeks carries additional risk, because it may predispose the child to disabling long-term consequences, even though the eye may be preserved. Preterm delivery should be considered only after weighing the risks and benefits and after consulting with the family, treating obstetrician, and neonatologist. If no tumors are found during prenatal screening, as in our case, then screening should be started immediately after birth and should continue on a weekly basis until 2 months of age. We found tumors at day 10 after birth, while Xu and colleagues11 found development of the first tumors at 7 weeks after birth. Frequent screening is recommended until age 28 months and if tumors are discovered, then follow up is recommended until the age of 7 years.11 In the current study we aimed to apply NGS-based technology for detection of RB1 gene mutations in a family with inherited retinoblastoma and then applied this technology for
Downloaded by [University of California, San Diego] at 21:10 26 February 2016
4
P. K. SHAH ET AL.
prenatal testing in the same family. Molecular approaches using microsatellite-based methods have been reported previously.5 With advanced molecular methods, accurate and comprehensive evaluation capable of detecting mutations ranging from large exon deletions to small point mutations is emerging. Blood sampling for RB1 gene mutation testing is performed for analysis of germline mutations. In our case the fetal DNA was isolated through amniocentesis which was positive for the same RB1 mutation in a heterozygous state. The mutation c.1281delA has been previously reported as a germline mutation in an isolated case of retinoblastoma (LOVD database id: RB1_01952). As the family wanted to continue with the pregnancy, serial 3-D ultrasound was performed every 2 weeks during the last trimester. Since no ocular abnormalities were detected, the pregnancy was carried on until term and on a meticulous post-delivery screening, we identified the tumors at an early stage and initiated successful treatment with TTT. Since the fovea was not involved in either eye, the child is expected to have good vision. Genetic counseling has a significant impact upon families with a history of retinoblastoma. It is important to understand the genetic implications for members of these families. Identification of disease segregating mutations in such families can potentially provide the advantage of eliminating frequent surveillance for siblings and offspring of the affected proband. Here we present one of the first cases of prenatal genetic diagnosis for retinoblastoma from India using a NGS platform. In the current study, the pedigree shows a positive family history of retinoblastoma on the maternal side (mother, maternal aunt), the proband (III1) and a history of spontaneous abortion in the proband’s sibling. This history prompted referral for genetic diagnosis as the parents were interested in understanding the risk for retinoblastoma in the developing fetus. Prenatal diagnosis showed a similar mutation to that of the affected proband (III1). This result offered the benefit of effective disease management to the child. Clinical examination of the proband’s mother and maternal grandmother revealed retinocytoma, which emphasizes the need for careful examination in relatives of retinoblastoma patients. Analysis and identification of individuals who do not harbor mutations eliminates the need for frequent surveillance, and in turn can reduce economic strain and psychological duress in family members.
Acknowledgements The authors would like to acknowledge the contribution made by Dr. Sujatha Jagadeesh, Mediscan, Chennai, and the Centre for Cellular and Molecular Biology (CCMB), Hyderabad, India, in isolating the fetal DNA.
Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the writing and content of this article.
References 1. Pandey AN. Retinoblastoma: An overview. Saudi J Ophthalmol 2014;28:310–315. 2. Shah PK, Narendran V, Kalpana N. Outcomes of intra- and extraocular retinoblastomas from a single institute in South India. Ophthalmic Genet 2015;36:248–250. 3. Lau CS, Choy KW, Fan DS, et al. Prenatal screening for retinoblastoma in Hong Kong. Hong Kong Med J 2008;14:391–394. 4. Junien C, Despoisse S, Turleau C, et al. Retinoblastoma, deletion 13q14, and esterase D: Application of gene dosage effect to prenatal diagnosis. Cancer Genet Cytogenet 1982;6:281–287. 5. Ramprasad VL, Madhavan J, Murugan S, et al. Retinoblastoma in India: microsatellite analysis and its application in genetic counseling. Mol Diagn Ther 2007;11:63–70. 6. Usmanov RH, Kivela T. Predicted trends in the incidence of retinoblastoma in the Asia-Pacific Region. Asia Pac J of Ophthalmol 2014;3:151–157. 7. Murphree LA, Chantada G. Staging & grouping of retinoblastoma. In: Singh AD, Damato BE, Peer J, Murphree LA, Perry JD, editors. Clinical ophthalmic oncology. Philadelphia, PA: Elsevier, 2007. pp 422–427. 8. Munier FL. Classification and management of seeds in retinoblastoma. Ellsworth Lecture Ghent 24 August 2013. Ophthalmic Genet 2014;35:193–207. 9. Paquette LB, Miller D, Jackson HA, et al. In utero detection of retinoblastoma with fetal magnetic resonance and ultrasound: Initial experience. AJP Rep 2012;2:55–62. 10. Manjandavida FP, Honavar SG, Reddy VA, et al. Management and outcome of retinoblastoma with vitreous seeds. Ophthalmology 2014;121:517–524. 11. Xu K, Rosenwaks Z, Beaverson K, et al. Preimplantation genetic diagnosis for retinoblastoma: The first reported liveborn. Am J Ophthalmol 2004;137:18–23. 12. Dhanjal S, Kakourou G, Mamas T, et al. Preimplantation genetic diagnosis for retinoblastoma predisposition. Br J Ophthalmol 2007;91:1090–1091.
ISSN: 1381-6810 (Print) 1744-5094 (Online) Journal homepage: http://www.tandfonline.com/loi/iopg20
Prenatal genetic diagnosis of retinoblastoma and report of RB1 gene mutation from India Parag K. Shah, S. Sripriya, V. Narendran & A. J. Pandian To cite this article: Parag K. Shah, S. Sripriya, V. Narendran & A. J. Pandian (2016): Prenatal genetic diagnosis of retinoblastoma and report of RB1 gene mutation from India, Ophthalmic Genetics, DOI: 10.3109/13816810.2015.1107595 To link to this article: http://dx.doi.org/10.3109/13816810.2015.1107595
Published online: 25 Feb 2016.
Submit your article to this journal
View related articles
View Crossmark data
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=iopg20 Download by: [University of California, San Diego]
Date: 26 February 2016, At: 21:10
OPHTHALMIC GENETICS http://dx.doi.org/10.3109/13816810.2015.1107595
CASE REPORT
Prenatal genetic diagnosis of retinoblastoma and report of RB1 gene mutation from India Parag K. Shaha, S. Sripriyab, V. Narendrana, and A. J. Pandianb
Downloaded by [University of California, San Diego] at 21:10 26 February 2016
a Department of Pediatric Retina & Ocular Oncology, Aravind Eye Hospital & Postgraduate Institute of Ophthalmology, Coimbatore, Tamilnadu, India; bSankara Nethralaya ONGC Department of Genetics and Molecular Biology, Vision Research Foundation, Chennai, Tamilnadu, India
ABSTRACT
ARTICLE HISTORY
Background: Retinoblastoma is the most common intraocular malignancy of childhood. There is a paucity of genetic testing and prenatal genetic diagnosis from India, which has the highest incidence worldwide. Materials and methods: RB1 gene screening of an 8-month-old female child with bilateral retinoblastoma was accomplished using next generation sequencing. The results were used for prenatal testing in this family. Results: A heterozygous germline mutation (chr13: 48951119delA; c.1281delA) was detected, which resulted in premature termination of a protein product (p.Glu428Argfs*29). Prenatal testing in maternal DNA revealed carrier status of the mother. Further clinical examination in the family members revealed retinocytomas in both eyes of the mother and maternal grandmother. Prenatal genetic testing of the developing fetus showed positivity for the mutation. As the family preferred to continue the pregnancy, serial 3-D ultrasounds were carried out every 2 weeks in the third trimester. Ten days after delivery, small extrafoveal tumors developed in both eyes, which were then treated successfully with transpupillary thermotherapy. Conclusion: We report the significance of genetic testing in the early detection and management of retinoblastoma from India.
Received 6 March 2015 Revised 27 September 2015 Accepted 3 October 2015
Introduction Retinoblastoma is a genetic disease caused by mutation in the RB1 gene. More than 90% of cases are sporadic while less than 10% are familial.1 Approximately 40% of cases have germline mutations, while the remaining 60% have somatic mutations.2 Prenatal genetic diagnosis of retinoblastoma has been reported previously.3,4 There are very few reports from India,5 which has the highest incidence of retinoblastoma in the world.6 We report one of the first cases of prenatal genetic diagnosis from India.
Case report An 8-month-old female child was brought to our hospital with complaints of squinting of eyes since birth. Pedigree analysis revealed a history of an eye tumor, likely retinoblastoma and childhood death of a maternal aunt. The child was able to fix and follow light with both eyes open. She resisted occlusion of the right eye. Anterior segment examination showed mild esotropia along with nystagmus in both eyes. Fundus examination of the right eye under anaesthesia showed a large whitish mass lesion involving the entire macular region with surrounding exudative retinal detachment and diffuse subretinal seeds. The left eye fundus showed total exudative detachment with a large subretinal mass (Figure 1). Ultrasonography of both eyes showed large mass lesions with intralesional calcification. Magnetic resonance
KEYWORDS
India; prenatal; RB1 gene; retinoblastoma
imaging of the brain showed no extraocular spread. A diagnosis of bilateral retinoblastoma (group D in the right eye and group E in the left eye) was made, according to the International Retinoblastoma Classification.7 The child was given six cycles of systemic chemotherapy using carboplatin, vincristine and etoposide, along with three injections of subTenon’s carboplatin to the right eye and transpupillary thermotherapy (TTT) to both eyes. At the end of the chemotherapy cycles, the tumors of both eyes showed type 3 regression (which is partial calcification with cottage cheese appearance of the tumor) while the vitreous seeds were calcified suggestive of type 1 vitreous seeds regression (which is refringent and/or calcified vitreous seeds residues).8 However 10 weeks later, tumor recurrence was observed in both eyes and as parents refused enucleation of the left eye, external beam radiotherapy (EBRT) was given at a dose of 36 Gy to both eyes. Following EBRT, both eyes showed good regression. Fundus examination of the mother showed large peripheral chorioretinal lesions with foci of calcification in both the eyes, suggestive of presumed retinocytomas (Figure 2). Fundus examination of the maternal grandmother of the proband also showed presumed retinocytomas in both eyes (Figure 3). RB1 gene screening was performed by Next Generation Sequencing (NGS) using the MiSeqIllumina NGS platform (Strand Life Science Pvt Ltd, Bangalore, India). Variations were identified by using the AVADIS® NGS software and interpreted using the StrandOmics tool. The observed variation was further validated by direct sequencing
CONTACT Parag K. Shah [email protected] Department of Pediatric Retina & Ocular Oncology, Aravind Eye Hospital & Postgraduate Institute of Ophthalmology, Avinashi Road, Coimbatore, Tamilnadu 641014, India. Color versions of one or more figures in the article can be found online at http://www.tandfonline.com/iopg. © 2016 Taylor & Francis
2
P. K. SHAH ET AL.
Downloaded by [University of California, San Diego] at 21:10 26 February 2016
Figure 1. (A) Retcam picture of the right eye showing a large macular retinoblastoma. (B) Left eye showing total exudative retinal detachment with a subretinal mass.
Figure 2. (A) Fundus picture of the right eye of the mother showing para macular flat lesions with calcification (white arrow) suggestive of retinocytoma. (B) Fundus picture of left eye showing retinocytoma (white arrow) in the temporal periphery.
Figure 3. (A) Fundus picture of the right eye of the grandmother showing a large retinocytoma involving the macula (white arrow). (B) Fundus picture of the left eye showing retinocytoma (white arrow) in the temporal periphery.
and cosegregation analysis was performed in the pedigree members. Next generation sequencing analysis of the RB1 gene in the illumina platform revealed a heterozygous deletion (chr13: 48951119delA c.1281delA) that resulted in the premature truncation of the retinoblastoma protein (lu428Argfs*29). Analysis of the mother’s DNA revealed the same heterozygous mutation and the father was normal (Figure 4). As the family wanted to have another child, after genetic counseling, prenatal diagnosis was offered. The fetal blood sample was collected by amniocentesis and tested for the specific known mutation by direct sequencing. Multi loci short tandem repeats (STR) analysis was performed to rule out maternal contamination. The fetal DNA was positive for the mutation in the heterozygous state. As they wanted to continue the pregnancy, serial 3-D ultrasounds were performed every 2 weeks during the third trimester.9 No eye abnormalities were observed on prenatal ultrasound imaging and child was delivered at full term. Fundus examination 3
days after birth revealed multiple birth-related retinal hemorrhages. No tumors were seen. Repeat examination at 10 days showed three new, small tumors in the right eye and two new tumors in the left eye (Figure 5). All were less than 3 mm in size. As none involved the fovea, immediate TTT was performed. Following this treatment, all the tumors showed a flat scar until the last follow up at 3 months.
Discussion India has the highest reported incidence of retinoblastoma.6 Of the predicted global burden of 8099 cases of retinoblastoma in 2013, India had 1486 cases, followed by China with 1103 cases.6 Although the survival rates in India have greatly increased,2,10 there is a paucity of genetic testing. With increased survival, we now see second and even third generations of children born to survivors. The potential benefits of genetic counseling and prenatal testing for retinoblastoma has
Downloaded by [University of California, San Diego] at 21:10 26 February 2016
OPHTHALMIC GENETICS
3
Figure 4. Cosegregation analysis of the c.1281delA mutation in exon 13 of the RB1 gene. Samples II1, III1 and III2 are positive for the nucleotide variation.
Figure 5. (A & B) Retcam fundus pictures of the right and left eyes at 10 days after birth showing small, round, creamy masses (white arrows) consistent with retinoblastoma.
previously been reported in a handful of cases in India.5With increased availability and awareness of this testing, prenatal genetic diagnosis is being used with increased frequency.2 Indications for prenatal screening include: the presence of bilateral retinoblastoma in either parent, retinocytoma in either parent (as in our case), or a positive family history of retinoblastoma. Methods of prenatal screening are preimplantation genetic diagnosis (PGD),11,12 prenatal genetic testing of the fetus, prenatal ultrasound, and fetal magnetic resonance imaging (MRI).9 PGD is done on 12-day-old embryos after in vitro fertilization. Unaffected embryos are then transferred to the uterus to continue the pregnancy. PGD although sensitive, is technically challenging and limited only to certain medical centers in the world. Both prenatal ultrasound and fetal MRI have limitations, in that they may not detect minimally elevated retinoblastoma tumors. Prenatal ultrasound is started between 16 and 18 weeks gestation, and repeated every 4 weeks until 32 weeks, then every 2 weeks until delivery. Fetal MRIs are performed every 8 weeks starting at 16–18 weeks of gestation.9 Compared to fetal MRI, prenatal
ultrasound is more sensitive for the detection of retinoblastoma tumors.9 If tumors are detected prenatally, then preterm delivery can be considered as an option, so that immediate treatment can be initiated. Delivery before 34 weeks carries additional risk, because it may predispose the child to disabling long-term consequences, even though the eye may be preserved. Preterm delivery should be considered only after weighing the risks and benefits and after consulting with the family, treating obstetrician, and neonatologist. If no tumors are found during prenatal screening, as in our case, then screening should be started immediately after birth and should continue on a weekly basis until 2 months of age. We found tumors at day 10 after birth, while Xu and colleagues11 found development of the first tumors at 7 weeks after birth. Frequent screening is recommended until age 28 months and if tumors are discovered, then follow up is recommended until the age of 7 years.11 In the current study we aimed to apply NGS-based technology for detection of RB1 gene mutations in a family with inherited retinoblastoma and then applied this technology for
Downloaded by [University of California, San Diego] at 21:10 26 February 2016
4
P. K. SHAH ET AL.
prenatal testing in the same family. Molecular approaches using microsatellite-based methods have been reported previously.5 With advanced molecular methods, accurate and comprehensive evaluation capable of detecting mutations ranging from large exon deletions to small point mutations is emerging. Blood sampling for RB1 gene mutation testing is performed for analysis of germline mutations. In our case the fetal DNA was isolated through amniocentesis which was positive for the same RB1 mutation in a heterozygous state. The mutation c.1281delA has been previously reported as a germline mutation in an isolated case of retinoblastoma (LOVD database id: RB1_01952). As the family wanted to continue with the pregnancy, serial 3-D ultrasound was performed every 2 weeks during the last trimester. Since no ocular abnormalities were detected, the pregnancy was carried on until term and on a meticulous post-delivery screening, we identified the tumors at an early stage and initiated successful treatment with TTT. Since the fovea was not involved in either eye, the child is expected to have good vision. Genetic counseling has a significant impact upon families with a history of retinoblastoma. It is important to understand the genetic implications for members of these families. Identification of disease segregating mutations in such families can potentially provide the advantage of eliminating frequent surveillance for siblings and offspring of the affected proband. Here we present one of the first cases of prenatal genetic diagnosis for retinoblastoma from India using a NGS platform. In the current study, the pedigree shows a positive family history of retinoblastoma on the maternal side (mother, maternal aunt), the proband (III1) and a history of spontaneous abortion in the proband’s sibling. This history prompted referral for genetic diagnosis as the parents were interested in understanding the risk for retinoblastoma in the developing fetus. Prenatal diagnosis showed a similar mutation to that of the affected proband (III1). This result offered the benefit of effective disease management to the child. Clinical examination of the proband’s mother and maternal grandmother revealed retinocytoma, which emphasizes the need for careful examination in relatives of retinoblastoma patients. Analysis and identification of individuals who do not harbor mutations eliminates the need for frequent surveillance, and in turn can reduce economic strain and psychological duress in family members.
Acknowledgements The authors would like to acknowledge the contribution made by Dr. Sujatha Jagadeesh, Mediscan, Chennai, and the Centre for Cellular and Molecular Biology (CCMB), Hyderabad, India, in isolating the fetal DNA.
Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the writing and content of this article.
References 1. Pandey AN. Retinoblastoma: An overview. Saudi J Ophthalmol 2014;28:310–315. 2. Shah PK, Narendran V, Kalpana N. Outcomes of intra- and extraocular retinoblastomas from a single institute in South India. Ophthalmic Genet 2015;36:248–250. 3. Lau CS, Choy KW, Fan DS, et al. Prenatal screening for retinoblastoma in Hong Kong. Hong Kong Med J 2008;14:391–394. 4. Junien C, Despoisse S, Turleau C, et al. Retinoblastoma, deletion 13q14, and esterase D: Application of gene dosage effect to prenatal diagnosis. Cancer Genet Cytogenet 1982;6:281–287. 5. Ramprasad VL, Madhavan J, Murugan S, et al. Retinoblastoma in India: microsatellite analysis and its application in genetic counseling. Mol Diagn Ther 2007;11:63–70. 6. Usmanov RH, Kivela T. Predicted trends in the incidence of retinoblastoma in the Asia-Pacific Region. Asia Pac J of Ophthalmol 2014;3:151–157. 7. Murphree LA, Chantada G. Staging & grouping of retinoblastoma. In: Singh AD, Damato BE, Peer J, Murphree LA, Perry JD, editors. Clinical ophthalmic oncology. Philadelphia, PA: Elsevier, 2007. pp 422–427. 8. Munier FL. Classification and management of seeds in retinoblastoma. Ellsworth Lecture Ghent 24 August 2013. Ophthalmic Genet 2014;35:193–207. 9. Paquette LB, Miller D, Jackson HA, et al. In utero detection of retinoblastoma with fetal magnetic resonance and ultrasound: Initial experience. AJP Rep 2012;2:55–62. 10. Manjandavida FP, Honavar SG, Reddy VA, et al. Management and outcome of retinoblastoma with vitreous seeds. Ophthalmology 2014;121:517–524. 11. Xu K, Rosenwaks Z, Beaverson K, et al. Preimplantation genetic diagnosis for retinoblastoma: The first reported liveborn. Am J Ophthalmol 2004;137:18–23. 12. Dhanjal S, Kakourou G, Mamas T, et al. Preimplantation genetic diagnosis for retinoblastoma predisposition. Br J Ophthalmol 2007;91:1090–1091.
