Вопросы гематологии/онкологии и иммунопатологии в педиатрии. 2022; 21: 78-88
Первые результаты проведения генетического скрининга и изучения генотип-фенотипических корреляций у пациентов с ретинобластомой из Беларуси
https://doi.org/10.24287/1726-1708-2022-21-2-78-88Аннотация
Ретинобластома – это агрессивное онкологическое заболевание глаза, происходящее из клеток-предшественников фоторецепторов сетчатки, наиболее часто дебютирующее в детском возрасте. У 98 % пациентов ретинобластома инициируется биаллельной инактивацией гена RB1, играющего важную роль в регуляции клеточного цикла и поддержании стабильности генома. Около 40 % нарушений в гене RB1 являются герминальными.
Цель – провести анализ частоты герминальных нарушений гена RB1 и их связи с клиническим фенотипом в группе белорусских пациентов с ретинобластомой. Данное исследование одобрено независимым этическим комитетом и утверждено решением ученого совета ГУ «Республиканский научно-практический центр детской онкологии, гематологии и иммунологии» (Республика Беларусь). В исследование включены 20 пациентов из неродственных семей (9 – с монолатеральной ретинобластомой, 11 – с билатеральной). У 2 из 11 пациентов с билатеральной ретинобластомой отмечен семейный анамнез заболевания. Геномную ДНК выделяли из суспензии лейкоцитов периферической крови. С полученной ДНК ставили серию полимеразных цепных реакций для амплификации фрагментов, включающих последовательности всех экзонов, регионы сплайс-сайтов и промоторные области. Детектирование нуклеотидных последовательностей полученных ампликонов выполняли методом высокопроизводительного секвенирования. Наличие клинически значимых нарушений подтверждали методом автоматического секвенирования по Сэнгеру. Крупные поломки определяли методом мультиплексной пробазависимой лигазной реакции (MLPA) или флуоресцентной гибридизации in situ (FISH). При выявлении нарушения у пробанда выполняли генетическое исследование его кровным родственникам (обследованы 5 семей). У 14 пациентов выявлено 13 различных генетических нарушений, 4 из которых ранее не были идентифицированы у пациентов с ретинобластомой среди других популяций (экзон 3: c.350_351delTT, p.Phe117TyrfsTer2; экзон 8: c.861+2T>G; экзон 24: c.2520+4A>G; делеция экзонов 16, 17). Все нарушения детектированы в гетерозиготном состоянии. В зависимости от типа генетические нарушения распределились следующим образом: 38,5 % (n = 5) дефектов в сплайс-сайтах; 15,4 % (n = 2) миссенс; 15,4 % (n = 2) небольших делеций, приводящих к сдвигу рамки считывания; 23% (n = 3) крупных делеций; 7,7 % (n = 1) нонсенс. Герминальные нарушения определены в 33,3 % (3/9) случаев с монолатеральной ретинобластомой и в 100 % (11/11) – с билатеральной. У 10 % пациентов определен наследственный характер ретинобластомы. При обследовании 5 семей у 3 пробандов выявленные нарушения определены как de novo. В статье представлены первые данные, полученные при генетическом исследовании белорусских пациентов с ретинобластомой. Методом секвенирования детектированы 78,6 % нарушений, тогда как методами MLPA и FISH – 21,4 %, что демонстрирует необходимость применения комплексного подхода. Среди спорадических нарушений в 66,6 % (12/18) случаев была затронута герминальная линия, что подчеркивает важность использования генетического тестирования при диагностике и клиническом мониторинге пациентов с ретинобластомой.
Список литературы
1. Liu J., Ottaviani D., Sefta M. A high-risk retinoblastoma subtype with stemness features, dedifferentiated cone states and neuronal / ganglion cell gene expression. Nat Commun 2021; 12 (1): 1–20. DOI: 10.1038/s41467-021-25792-0
2. OMIM Entry – #180200 – Retinoblastoma; RB1 [Electronic resource]. Available at: https://www.omim.org/entry/180200?search=R-b1&highlight=rb1 (accessed December 25, 2021).
3. Bornfeld N., Lohmann D., Bechrakis N. Retinoblastom. Ophthalmologe 2020; 117 (4): 389–402. DOI: 10.1007/s00347-020-01081-x
4. Alkatan H. M., Al Marek F., Elkhamary S. Demographics of pediatric orbital lesions: A tertiary eye center experience in Saudi Arabia. J Epidemiol Glob Health 2019; 9 (1): 3–10. DOI: 10.2991/jegh.k.181224.001
5. Liu Z., Yang Q., Cai N. Enigmatic differences by sex in cancer incidence: evidence from childhood cancers. Am J Epidemiol 2019; 188 (6): 1130–5. DOI: 10.1093/aje/kwz058
6. Tomar A. S., Finger P. T., Gallie B. Global retinoblastoma treatment outcomes: association with national income level. Ophthalmology 2021; 128 (5): 740–53. DOI: 10.1016/j.ophtha.2020.09.032
7. Fabian I. D., Abdallah E., Abdullahi S. U. Global retinoblastoma presentation and analysis by national income level. JAMA Oncol 2020; 6 (5): 685–95. DOI: 10.1001/jamaoncol.2019.6716
8. Ensembl genome browser 104 [Electronic resource]. Ensembl release 97. Available at: http://www.ensembl.org/index.html (accessed December 28, 2021).
9. Zou Y., Li J., Hua P. Spectrum of germline mutations in RB1 in Chinese patients with retinoblastoma: Application of targeted next-generation sequencing. Mol Vis 2021; 27: 1–16.
10. Kooi I. E., Mol B. M., Massink M. P. G. Somatic genomic alterations in retinoblastoma beyond RB1 are rare and limited to copy number changes. Sci Rep 2016; 6 (1): 1–11. DOI: 10.1038/srep25264
11. Francis J. H., Richards A. L., Mandelker D. L. Molecular changes in retinoblastoma beyond RB1: Findings from next-generation sequencing. Cancers (Basel) 2021; 13 (1): 149–61. DOI: 10.3390/cancers13010149
12. Nummi K., Kivelä T. Retinoblastoma in Finland, 1964–2014: incidence and survival. Br J Ophthalmol 2021; 105 (1): 63–9. DOI: 10.1136/bjophthalmol-2019-315744
13. Munier F., Beck-Popovich M., Chantada G. Concervative management of retinoblastoma: challenging orthodoxy without compromising the state of metastatic grace. “Alive, with good vision no comorbidity”. Prog Retin Eye Res 2020; 73: 5. DOI: 10.1016/j.preteyeres.2019.05.005
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15. Sheehan A. P. Retinoblastoma: Early diagnosis is crucial. J Pediatr Health Care 2020; 34 (6): 601–5. DOI: 10.1016/j.pedhc.2020.06.006
16. Алексеева Е. А. Эффект родительского происхождения мутации в гене RB1 при наследственной ретинобластоме с низкой пенетрантностью / Е. А. Алексеева [и др.] // Медицинская генетика. – 2019. – 18 (8): 21–8. URL: https://www.medgen-journal.ru/jour/article/viewFile/714/439
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21. Skalet A. H., Gombos D. S., Gallie B. L. Screening children at risk for retinoblastoma: Consensus report from the American Association of Ophthalmic Oncologists and Pathologists. Ophthalmology 2018; 125 (3): 453–8. DOI: 10.1016/j.ophtha.2017.09.001
22. Garza-Garza L. A., Ruiz-Lozano R. E., Rebolledo-Méndez G. Challenge of Retinoblastoma in Mexico in 2020: Perspectives and Solutions. J Ophthalmol 2020; 2020: 1953602. DOI: 10.1155/2020/1953602
23. Knudson A. G. Mutation and Cancer: Statistical Study of Retinoblastoma. Proc Natl Acad Sci U S A 1971; 68 (4): 820–3. DOI: 10.1073/pnas.68.4.820
24. Mendoza P. R., Grossniklaus H. E. The Biology of Retinoblastoma. Prog Mol Biol Transl Sci 2015; 134: 503–16. DOI: 10.1016/bs.pmbts.2015.06.012
25. McGowanJordan J., Simons A., Schmid M. An International System for Human Cytogenetic Nomenclature (1985) ISCN 1985. Report of the Standing Committee on Human Cytogenetic Nomenclature. Birth Defects Orig Artic Ser 1985; 21 (1): 1–117.
26. HGMD (The Human Gene Mutation Database) [Electronic resource]. Available at: https://genschool.ru/wa-data/public/site/1.4_Mutatsii_Strelnikov_VV.pdf (accessed December 06, 2021).
27. LOVD (Leiden Open Variation Data-base Online genecentered collection and display of DNA variants) [Electronic resource]. Available at: https://www.lovd.nl/ (accessed December 06, 2021).
28. Valverde J. R., Alonso J., Palacios I. RB1 gene mutation up-date, a meta-analysis based on 932 reported mutations available in a searchable database. BMC Genet. 2005; 6: 53. DOI: 10.1186/1471-2156-6-53
29. Dommering C. J., Mol B. M., Moll A. C. RB1 mutation spectrum in a comprehensive nationwide cohort of retinoblastoma patients. J Med Genet 2014; 51 (6): 366–74. DOI: 10.1136/jmedgenet-2014-102264
30. Mehyar M., Mosallam M., Tbakhi A. Impact of RB1 gene mutation type in retinoblastoma patients on clinical presentation and management outcome. Hematol Oncol Stem Cell Ther 2020; 13 (3): 152–9. DOI: 10.1016/j.hemonc.2020.02.006
31. Tomar S., Sethi R., Sundar G., Quah T. C., Quah B. L., Lai P. S. Mutation spectrum of RB1 mutations in retinoblastoma cases from Singapore with implications for genetic management and counselling. PloS One, 2017; 12 (6): 1–23. DOI: 10.1371/journal.pone.0178776
32. Zhang Z., Xiao Y., Shen R. Next generation sequencing of RB1gene for the molecular diagnosis of ethnic minority with retinoblastoma in Yunnan. BMC Med Genet 2020; 21 (1): 230. DOI: 10.1186/s12881-020-01150-7
33. Kiet N. C., Khuong L. T., Minh D. D. Spectrum of mutations in the RB1 gene in Vietnamese patients with retinoblastoma. Mol Vis 2019; 25: 215–21.
34. Soliman S. E., Racher H., Zhang C. Genetics and Molecular Diagnostics in Retinoblastoma--An Update. Asia Pac J Ophthalmol (Phila) 2017; 6 (2): 197–207. DOI: 10.22608/APO.201711
35. Rojanaporn D., Boontawon T., Chareonsirisuthigul T. Spectrum of germline RB1 mutations and clinical manifestations in retinoblastoma patients from Thailand. Mol Vis 2018; 24: 778–88.
Pediatric Hematology/Oncology and Immunopathology. 2022; 21: 78-88
The first results of genetic screening and exploration of genotype-phenotype correlations in retinoblastoma patients from Belarus
https://doi.org/10.24287/1726-1708-2022-21-2-78-88Abstract
Retinoblastoma is an aggressive eye tumor originating from maturing cone precursors in the developing retina and most commonly seen in childhood. In 98 % of patients, retinoblastoma is caused by bi-allelic inactivation of the RB1 tumor suppressor gene. Approximately 40 % of disorders in the RB1 gene are germline.
This study aimed to analyze the frequency of germline RB1 variants in a cohort of Belarusian patients with retinoblastoma and to correlate the variants with clinical phenotypes. The study was approved by an Independent Ethics Committee and the Scientific Council of the Belarusian Research Center for Pediatric Oncology, Hematology and Immunology. The study included 20 patients from unrelated families (9 patients with unilateral retinoblastoma, 11 – with bilateral). Two out of eleven patients with bilateral retinoblastoma had a positive family history. Genomic DNA was extracted from peripheral blood mononuclear cells. Using polymerase chain reaction, we obtained fragments including sequences of all exons, regions of splice sites and promoter regions of the RB1 gene. Nucleotide sequences of the obtained amplicons were detected by next-generation sequencing. All clinically significant variants were confirmed by Sanger sequencing. Multiplex ligation-dependent probe amplification (MLPA) or fluorescence in situ hybridization (FISH) were used to detect gross alterations. A genetic analysis of blood relatives was carried out for five probands with detected germline variants. We identified 13 different variants in 14 patients: 38.5 % (n = 5) of them were defects in splice sites; 15.4 % (n = 2) – missense mutations; 15.4 % (n = 2) – small deletions (frameshift); 23% (n = 3) – large deletions; 7.7% (n = 1) – nonsense mutations. Four of these variants had not been previously reported in patients with retinoblastoma from other populations (exon 3: c.350_351delTT, p. Phe117TyrfsTer2; exon 8: c.861+2T>G; exon 24: c.2520+4A>G; Del of exons 16, 17). Germline mutations were detected in 33.3 % (3/9) of patients with unilateral retinoblastoma and in 100% (11/11) of patients with bilateral disease. A genetic screening of relatives showed that three variants were de novo, and two variants were inherited from parents in families with a positive history of retinoblastoma. Here we reported the first results of genetic examination of Belarusian patients with retinoblastoma. Seventy-eight point six per cent (78.6 %) of variants were detected by sequencing, 21.4 % were identified with the help of the MLPA and FISH methods. Among sporadic cases, germline RB1 variants were detected in 66.6 % (12/18) of cases. A full range of screening techniques is required to achieve high sensitivity of detection in retinoblastoma patients. Our study also provides new evidence that will inform patient management and genetic counseling.
References
1. Liu J., Ottaviani D., Sefta M. A high-risk retinoblastoma subtype with stemness features, dedifferentiated cone states and neuronal / ganglion cell gene expression. Nat Commun 2021; 12 (1): 1–20. DOI: 10.1038/s41467-021-25792-0
2. OMIM Entry – #180200 – Retinoblastoma; RB1 [Electronic resource]. Available at: https://www.omim.org/entry/180200?search=R-b1&highlight=rb1 (accessed December 25, 2021).
3. Bornfeld N., Lohmann D., Bechrakis N. Retinoblastom. Ophthalmologe 2020; 117 (4): 389–402. DOI: 10.1007/s00347-020-01081-x
4. Alkatan H. M., Al Marek F., Elkhamary S. Demographics of pediatric orbital lesions: A tertiary eye center experience in Saudi Arabia. J Epidemiol Glob Health 2019; 9 (1): 3–10. DOI: 10.2991/jegh.k.181224.001
5. Liu Z., Yang Q., Cai N. Enigmatic differences by sex in cancer incidence: evidence from childhood cancers. Am J Epidemiol 2019; 188 (6): 1130–5. DOI: 10.1093/aje/kwz058
6. Tomar A. S., Finger P. T., Gallie B. Global retinoblastoma treatment outcomes: association with national income level. Ophthalmology 2021; 128 (5): 740–53. DOI: 10.1016/j.ophtha.2020.09.032
7. Fabian I. D., Abdallah E., Abdullahi S. U. Global retinoblastoma presentation and analysis by national income level. JAMA Oncol 2020; 6 (5): 685–95. DOI: 10.1001/jamaoncol.2019.6716
8. Ensembl genome browser 104 [Electronic resource]. Ensembl release 97. Available at: http://www.ensembl.org/index.html (accessed December 28, 2021).
9. Zou Y., Li J., Hua P. Spectrum of germline mutations in RB1 in Chinese patients with retinoblastoma: Application of targeted next-generation sequencing. Mol Vis 2021; 27: 1–16.
10. Kooi I. E., Mol B. M., Massink M. P. G. Somatic genomic alterations in retinoblastoma beyond RB1 are rare and limited to copy number changes. Sci Rep 2016; 6 (1): 1–11. DOI: 10.1038/srep25264
11. Francis J. H., Richards A. L., Mandelker D. L. Molecular changes in retinoblastoma beyond RB1: Findings from next-generation sequencing. Cancers (Basel) 2021; 13 (1): 149–61. DOI: 10.3390/cancers13010149
12. Nummi K., Kivelä T. Retinoblastoma in Finland, 1964–2014: incidence and survival. Br J Ophthalmol 2021; 105 (1): 63–9. DOI: 10.1136/bjophthalmol-2019-315744
13. Munier F., Beck-Popovich M., Chantada G. Concervative management of retinoblastoma: challenging orthodoxy without compromising the state of metastatic grace. “Alive, with good vision no comorbidity”. Prog Retin Eye Res 2020; 73: 5. DOI: 10.1016/j.preteyeres.2019.05.005
14. Ishaq H., Patel B. Retinoblastoma; Florida: StatPearl. Treasure Island; 2021 [Updated 2021 Aug 11]. [Electronic resource]. Available at:https://www.ncbi.nlm.nih.gov/books/NBK545276/ (accessed 06. 01. 2022).
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17. Balmer A., Zografos L., Munier F. Diagnosis and current management of retinoblastoma. Oncogene 2006; 25 (38): 5341–9. DOI: 10.1038/sj.onc.1209622
18. Gallie B., Ellsworth R., Abramson D., Phillips R. Retinoma: spontaneous regression of retinoblastoma or benign manifestation of the mutation? Br J Cancer 1982; 45 (4): 513–21.
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20. Mustafa M., Mohammad M., Abdelghani T. Impact of RB1 gene mutation type in retinoblastoma patients on clinical presentation and management outcome. Hematol Oncol Stem Cell Ther 2020; 13 (3): 152–9. DOI: 10.1016/j.hemonc.2020.02.006
21. Skalet A. H., Gombos D. S., Gallie B. L. Screening children at risk for retinoblastoma: Consensus report from the American Association of Ophthalmic Oncologists and Pathologists. Ophthalmology 2018; 125 (3): 453–8. DOI: 10.1016/j.ophtha.2017.09.001
22. Garza-Garza L. A., Ruiz-Lozano R. E., Rebolledo-Méndez G. Challenge of Retinoblastoma in Mexico in 2020: Perspectives and Solutions. J Ophthalmol 2020; 2020: 1953602. DOI: 10.1155/2020/1953602
23. Knudson A. G. Mutation and Cancer: Statistical Study of Retinoblastoma. Proc Natl Acad Sci U S A 1971; 68 (4): 820–3. DOI: 10.1073/pnas.68.4.820
24. Mendoza P. R., Grossniklaus H. E. The Biology of Retinoblastoma. Prog Mol Biol Transl Sci 2015; 134: 503–16. DOI: 10.1016/bs.pmbts.2015.06.012
25. McGowanJordan J., Simons A., Schmid M. An International System for Human Cytogenetic Nomenclature (1985) ISCN 1985. Report of the Standing Committee on Human Cytogenetic Nomenclature. Birth Defects Orig Artic Ser 1985; 21 (1): 1–117.
26. HGMD (The Human Gene Mutation Database) [Electronic resource]. Available at: https://genschool.ru/wa-data/public/site/1.4_Mutatsii_Strelnikov_VV.pdf (accessed December 06, 2021).
27. LOVD (Leiden Open Variation Data-base Online genecentered collection and display of DNA variants) [Electronic resource]. Available at: https://www.lovd.nl/ (accessed December 06, 2021).
28. Valverde J. R., Alonso J., Palacios I. RB1 gene mutation up-date, a meta-analysis based on 932 reported mutations available in a searchable database. BMC Genet. 2005; 6: 53. DOI: 10.1186/1471-2156-6-53
29. Dommering C. J., Mol B. M., Moll A. C. RB1 mutation spectrum in a comprehensive nationwide cohort of retinoblastoma patients. J Med Genet 2014; 51 (6): 366–74. DOI: 10.1136/jmedgenet-2014-102264
30. Mehyar M., Mosallam M., Tbakhi A. Impact of RB1 gene mutation type in retinoblastoma patients on clinical presentation and management outcome. Hematol Oncol Stem Cell Ther 2020; 13 (3): 152–9. DOI: 10.1016/j.hemonc.2020.02.006
31. Tomar S., Sethi R., Sundar G., Quah T. C., Quah B. L., Lai P. S. Mutation spectrum of RB1 mutations in retinoblastoma cases from Singapore with implications for genetic management and counselling. PloS One, 2017; 12 (6): 1–23. DOI: 10.1371/journal.pone.0178776
32. Zhang Z., Xiao Y., Shen R. Next generation sequencing of RB1gene for the molecular diagnosis of ethnic minority with retinoblastoma in Yunnan. BMC Med Genet 2020; 21 (1): 230. DOI: 10.1186/s12881-020-01150-7
33. Kiet N. C., Khuong L. T., Minh D. D. Spectrum of mutations in the RB1 gene in Vietnamese patients with retinoblastoma. Mol Vis 2019; 25: 215–21.
34. Soliman S. E., Racher H., Zhang C. Genetics and Molecular Diagnostics in Retinoblastoma--An Update. Asia Pac J Ophthalmol (Phila) 2017; 6 (2): 197–207. DOI: 10.22608/APO.201711
35. Rojanaporn D., Boontawon T., Chareonsirisuthigul T. Spectrum of germline RB1 mutations and clinical manifestations in retinoblastoma patients from Thailand. Mol Vis 2018; 24: 778–88.
