RB1 AND BEYOND: DETERMINING GENETIC CAUSES OF RETINOBLASTOMA IN SOUTH AFRICAN PATIENTS Danielle Beukman Student number: 719425 Supervisors: Dr. Lindie Lamola Ms. Nkateko Mayevu A research report in the format of a “submissible” paper submitted to the Faculty of Health Sciences, University of the Witwatersrand, Johannesburg. This research report is required for partial fulfilment of the requirements for the degree of Master of Science in Medicine Genomic Medicine Johannesburg, South Africa 25 February 2024 NATIONAL HEALTH LABORATORY SERVICE School of Pathology, University of the Witwatersrand DIVISION OF HUMAN GENETICS Hospital Street, Johannesburg, 2001 ǀ PO Box 1038, Johannesburg, 2000 [T] +27 11 489 9223 ǀ [M] +27 78 080 8841 ǀ [F] +27 11 489 9226 ǀ [E] human.genetics@nhls.ac.za 30 November 2023 Re: Research report submitted to the Faculty of Health Sciences by Ms. Danielle Beukman in partial fulfilment of the requirements for the degree of Master of Science in Medicine (Genomic Medicine) I would like to thank you for agreeing to examine my research report titled “RB1 and beyond: Determining genetic causes of retinoblastoma in South African patients” The candidate, Ms. Danielle Beukman, is submitting her research report in the format of a “submissible” format. The research report contributes 33% towards the final mark of the degree. The candidate and her supervisors have chosen to submit the paper to the journal “Human Genetics” published by Springer. The submissible paper attached is therefore written in accordance with the author guidelines of the above- mentioned journal. These guidelines have been attached as a website link for your reference. The only deviation from these guidelines is that the manuscript has been presented as one complete document (including tables, figures, and list of abbreviations) to make marking easier, rather than being submitted separately. The research protocol is attached as an appendix for the purposes of providing an extended literature review and further context to the study. The protocol has already been assessed and approved by the Faculty of Health Sciences and is therefore not for examination. Please refer to the Table of Contents for a complete list of the contents provided in this research report. Yours sincerely, Student: Ms. Danielle Beukman Supervisor: Dr Lindie Lamola Co-supervisor: Ms. Nkateko Mayevu This report is intended solely to record the observations and/or opinion of the writer. It does not constitute a medico-legal report mailto:human.genetics@nhls.ac.za i Declaration I, Danielle Beukman, declare that this research report, in the form of a “submissible” paper, is my own, unaided work. It has not been submitted before for any degree or examination at any other University. This research report is required for the Degree of Master of Science Genomic Medicine at the University of the Witwatersrand, Johannesburg. Danielle Beukman WITS student number 719425 Signature of candidate, Danielle Beukman Signed on 26 February 2024, in Johannesburg, RSA ii Contribu on of the candidate to the paper Declara on: Student’s contribu on to ar cle(s) and agreement of co-authors I, Danielle Beukman, student number 719425, declare that this Research Report, en tled “RB1 and Beyond: Determining gene c causes of Re noblastoma in South African pa ents” is my own work and that I made significant contribu ons towards the research findings presented in this paper, intended to be submi ed for publica on below. Signature of Student Date: 15 November 2023 Signature of Primary Supervisor Dr. L.Lamola Date: 16 November 2023 Agreement by co-authors By signing this declara on, the co-authors listed below agree to the use of the research fin d i ngs in tended for a published ar cle by a student as part of their Research Report. Authors Name Signature Date 1st Ms. Danielle Beukman 15 November 2023 2nd Dr. Lindiwe Lamola 16 November 2023 3rd Ms. Nkateko Mayevu 16 November 2023 iii Dedication I dedicate this research report to my family, in particular my beloved mother, Margaret and elder sister, Natasha. Your everlasting support has immeasurable value. iv Abstract Retinoblastoma is the most common solid tumour of the retina, affecting mainly paediatric patients. Although loss-of-function germline variants the RB1 gene is the tumour suppressor gene most often associated with heritable retinoblastoma, two contradictory research findings complicate the assumption that a pathogenic germline variant in RB1 will lead to the development of heritable retinoblastoma. Firstly, heritable retinoblastoma has been observed in cohorts with biallelic wild-type RB1 genes, secondly, biallelic loss-of-function is not always sufficient to lead to retinoblastoma tumorigenesis. By investigating germline variants in additional cancer predisposition genes to discover candidate driver genes in heritable retinoblastoma, the full germline mutational spectrum of heritable retinoblastoma can be established. In this study we investigate sequence germline variants in cancer predisposition genes beyond RB1 in South African retinoblastoma patients with an African ancestry by performing whole exome sequencing on eight patients diagnosed with retinoblastoma. Whole exome sequencing data for genes previously associated with retinoblastoma was subjected to variant annotation by means of a variant effect predictor and filtering criteria applied manually. Variant classification was performed according to guidelines proposed by The American College of Medical Genetics and Genomics and the Association of Molecular Pathology. The highest degree of variant classification was variants of unknown significance. The absence of sequence variants capable of describing the retinoblastoma phenotype in this cohort demonstrates the necessity of looking beyond RB1, combining next generation sequencing with copy number analysis pipelines and additional genome mapping technologies capable of detecting structural variants. The detection of sequence variants previously reported to be possibly damaging but now considered polymorphisms highlights the importance of revisiting variant classification. In closing, this study adds genomic information from patients with a South African ancestry to mounting genomic data, ensuring that previously underrepresented populations can also benefit from future cancer predisposition and precision medicine research. v Acknowledgements I would like to give special thanks to my supervisors, Dr. Lindie Lamola and Ms. Nkateko Mayevu. Your guidance, support and advice made me grow ever curious, but never defeated. I appreciate your patience and hard work. Thank you for allowing me to engage in the PeCan Project, an inspiring research venture that is now a cause close to my heart. Thank you to the WITS Division of Human Genetics academics. Professor Krause, Ms. Monica Araujo, Doctor L. Lamola, Doctor R. Kerr and Doctor Z. Lombard, and Doctor F. Baine- Savanhu, your passion for research and teaching is remarkable. Thank you all for the time you dedicated to us. Your enthusiasm for advancing genetic and genomic research in South Africa is infectious. I feel honored to have shared this year with my classmates, Anréé, Ingrid and Racilya, your friendship made every Wednesday worth it! I cherish every moment. Dylan, Thandeka, Phophi and Gabriella, thank you for being there when I needed advice. Most importantly, to God and my family, thank you for keeping the rent low and spirits high. To my mom Kleintjie, thank you for your unconditional support and the endless supply of Earl Grey tea. Dankie ma. vi Table of Contents Declaration…………………………………………………………………………………………………….……………………..i Contribution of the candidate to the paper …………………………………………………………..………..…….ii Dedication ………………………………………………………………………………………………………………..…………iii Abstract ………………………………………………………………………………………………………………………….……iv Acknowledgements ………………………………………………..……………………………………………..…….………v Table of Contents……………………………………………………………………………………………………………...….vi List of Appendices ……………….……………………………………………………………………………………..………vii List of Figures………………………………………………………………………………………………………………….….viii List of Tables…………………………………………………………………………………………………………………………ix Table of Abbreviations ..………………………………………………………………………………………………….…...x Research report in the form of a “submissible” paper………………………………………………………….xi Title page…………………………………………………………………………………….……………………………………….xi 1. Abstract for journal submission ……………………………………………………………………….………………1 2. Background………………………………………………………..………………………………………………………….…2 2.1. Retinoblastoma in South Africa……………………………………………….…………………………….2 2.2. Future implications of a definitive genetic diagnosis……………………………………………..2 2.3. The genetic landscape of RB….…………..…………………………………………………………………....3 2.4. Locus heterogeneity observed in RB predisposition……..………………………………………....4 3. Materials and Methods……..………..…………………………………………………………………………………..7 3.1. Ethical considerations and Consent ……………………………………………………………..…….7 3.2. Clinical Presentation and patient demographics………………………………………..….…….7 3.3. Whole exome sequencing ……………………………………………………………………………….…8 3.3.1. DNA sample preparation and sample quality control……………………………8 3.3.2. Library generation ……………………………………………………………………………….8 3.3.3. Templating and sequencing………………………………………………………………….8 3.4. Variant interpretation……………………………………………………………………..………………….9 4. Results …………………………………………………………………….……………………………………….……………10 4.1. Clinical presentation of patients…………………….…………….……………………………………10 4.2. Variant annotation…………………………………………..……………………………………………….10 4.3. RB1 sequence variants ……………………………………………………………..………………….…..11 4.4. Variants of uncertain significance …………………..…………………….………………………….11 4.5. Shared variants……………………………………………………………….………………………………..14 5. Discussion …………………………………………………….……………………………………………………..……..…15 5.1. RB1 variants ……………………………………………………………….………………………………..….15 5.2. Shared variants …………………………………………..……..………………………………………..….16 5.3. Variants of uncertain significance …………..……………..……………………………..………...17 5.4. Future prospects and limitations……………………………………………………………….………18 6. Conclusion ……………………………………………………………………………………………………………………..19 7. References…………………………..……………………………………………………………………………………..….20 Appendices…………………………………………………………………………………………………………………………29 vii List of Appendices Appendix A: Human Research Ethics Committee (Medical), University of the Witwatersrand Ethics Certificate (M180855) Appendix B: Human Research Ethics Committee (Medical), University of the Witwatersrand Ethics Certificate (M230782) Appendix C: RB1 sequence variants in all patients Appendix D Extended list of variants of interest Appendix E BAM images to evaluate variant coverage Appendix F: Information, Consent and Assent forms Appendix G: Title Approval and Approved Research Protocol Appendix H: Plagiarism declaration and Turnitin report Appendix I: Journal Author Guidelines for Submissible format viii List of Figures Figure 1: Distribution of annotated variants…………………………………………………..………………………………………………10 Figure 2: Distribution of variants in coding regions ……………………………………..…………………………………………………11 ix List of Tables Table 1: Genes implicated in the aetiology of RB development ………………………………………………………………………6 Table 2: Clinical presentation of RB patients…………………………………….………………………………………………………………7 Table 3: Subset of variants of interest as observed per patient ……….……………………………………………………………13 Table 4: Shared variants ………………………………………………………………………………………………………………………………14 x Table of Abbreviations Abbreviation Meaning ACMG/AMP American College of Medical Genetics and Genomics and Association for Molecular Pathology BAM Binary alignment map CADD Combined Annotation-Dependent Depletion CMA Chromosomal microarray CNV Copy number variation HREC Medical Human Research Ethics Committee (Medical) IGV Integrative genomics viewer Indels Insertions and Deletions LOVD Leiden Open Variation Database MAF Minor Allele Frequency miRNA Micro RNA MLPA Multiplex ligation-dependent probe amplification PeCan Paediatric Cancer POLYPHEN2 Polymorphism Phenotyping version 2 RB Retinoblastoma RB1 Retinoblastoma 1 tumor suppressor gene SNV Single Nucleotide Variants SIFT Sort Intolerable from Tolerable SANCR South African National Cancer Registry TSG Tumor suppressor gene VCF Variant Call files VEP Variant effect predictor VUS Variant of uncertain significance WES Whole exome sequencing xi Research report in the format of a “submissible” paper To be submitted to the Journal Human Mutation RB1 and Beyond: Determining genetic causes of Retinoblastoma in South African patients Danielle Beukman1, Nkateko Mayevu1, Lindie Lamola1 1 Division of Human Genetics, National Health Laboratory Service and School of Pathology, Faculty of Health Sciences, University of the Witwatersrand, JHB, South Africa Corresponding Author: Danielle Beukman Division of Human Genetics, Corner Hospital Street and De Korte Street, JHB, South Africa, 2001 [T]: 084 583 4985 [E]: beukmandanielle@gmail.com / 719425@students.wits.ac.za mailto:beukmandanielle@gmail.com mailto:719425@students.wits.ac.za 1 1. Abstract for Journal Submission Background: Retinoblastoma is the most common solid tumour of the retina, affecting mainly paediatric patients. Although loss-of-function germline variants the RB1 gene is the tumour suppressor gene most often associated with heritable retinoblastoma, two confounding research findings contradict the direct genotype phenotype relationship. First, heritable retinoblastoma has been observed in cohorts with biallelic wild-type RB1 genes, secondly, biallelic loss-of-function is not always sufficient to lead to retinoblastoma tumorigenesis. By investigating germline variants in additional cancer predisposition genes beyond RB1 allows for discovery of candidate driver genes in heritable retinoblastoma. In this study we investigate sequence germline variants in cancer predisposition genes beyond RB1 in retinoblastoma patients with a South African ancestry. Methods: Whole exome sequencing with single nucleotide resolution was performed to establish germline sequence variants. Variant annotation, prioritization and filtering was executed to detect germline variants in cancer predisposition genes previously proposed as candidate genes in retinoblastoma malignancy and paediatric cancer predisposition syndromes. Variant prioritization excluded alleles with a minor allele frequency above 5% and variants predicted to have no impact on a protein level due to no change in the amino acid sequence and protein structure. Variants shared between individuals and variants of interest were classified according to ACMG/AMP guidelines. Results: A total of 353 655 variant were annotated, 4538 of which were novel. After collating functional data, previously reported and reviewed RB1 variants, all sequence variants in RB1 are likely benign, (NM_000321.3:c.1574C>G, NP_000312.2:p.Ala525Gly and NM_000321.3:c.45_53del NP_000312.2:p.Ala16_Ala18del) amending outdated variants incorrectly classified as likely pathogenic. The highest degree of variant classification in genes previously associated with retinoblastoma are variants of unknown significance. Conclusion: The absence of sequence variants capable of describing the retinoblastoma phenotype in this cohort demonstrates the necessity of combining next generation sequencing with analysis pipelines and additional genome mapping to detect structural variants. This study adds genomic information from patients with a South African ancestry, an underrepresented population. Keywords: germline variation, cancer predisposition genes, whole exome sequencing, RB1 gene, retinoblastoma, sequence variants 2 2. Background 2.1. Retinoblastoma in South Africa Retinoblastoma (RB) is the most commonly occurring solid intraocular cancer, developing in childhood. The South African National Cancer Registry (SANCR) documented an average RB incidence of 1 in 21 641 live births for the time elapsed between 2004 and 2018 (https://www.nicd.ac.za/centres/national- cancer-registry). South African patients have a later mean age of diagnosis (2.5 years), compared to an earlier mean age of diagnosis of 1.2 years in high income countries (Stuart et al., 2022). Routine cancer surveillance is poor in South Africa due to an overburdened public health care system. As an early age of diagnosis is the strongest indicator of an improved prognosis, a later mean age of diagnosis puts South African children suffering from paediatric cancers in a disadvantageous position (Stuart et al., 2022). Recent efforts have been made to address South Africa’s later mean age of cancer diagnosis compared to global averages. The overall survival rate for RB patients in South Africa increased from 52% in 1994 (Hesseling et al., 1995) to 86.7% twenty years later (Ndlovu et al., 2023). Limiting disease for RB exhibits a 100% overall survival rate, emphasizing the importance of early detection. Children with a black South African ancestry have a disproportionately high incidence of RB compared to other racial groups (Ndlovu et al., 2023). The majority (60%) of RB cases are sporadic and unilateral (tumors affecting a single eye), occurring at a mean age of 24 months. The remaining 40% of RB cases are heritable, bilateral (tumors affecting both eyes) RB, with a significantly earlier mean age of onset of 12 months (Bouchoucha et al., 2023; Knudson, 1971). A small proportion of RB patients present with pineoblastoma in conjunction with bilateral RB, referred to as trilateral RB. The presence of a constitutional (germline) mutation in RB1 can either be inherited from a parent or occur de novo at an early stage of embryonic development, a mutation occurring at a later stage of embryonic development will result in mosaicism (Lin & Chintagumpala, 2021; Rushlow et al, 2009). 2.2. Future implications of a definitive genetic diagnosis The detection of a disease-causing germline variant and a mutation signature can aid in treatment planning and risk assessment. Patients with a pathogenic germline variant in RB1 have a predilection to developing further malignancies (Ketteler et al., 2020; Shinohara et al., 2014) and a recurrence risk 3 in offspring. A literature search into genetic variants predisposing black South African individuals to RB malignancy proved futile, revealing a gap in our understanding of hereditary and paediatric cancers in patients with a South African ancestry. Black South Africans are greatly underrepresented genetic research, the paucity of genomic sequencing data from black South African individuals creates a challenge when cancer screening and risk assessment protocols are designed. The aim of this study was to detect sequence variants predisposing paediatric black South African patients to developing RB. 2.3. The genetic landscape of RB The first tumor suppressor gene (TSG) described fully was the Retinoblastoma 1 (RB1) gene, by Alfred Knudson in 1971, a revolutionary finding that initiated exploration into cancer predisposition genes. More than a decade later, in 1986, the RB1 gene position was identified by positional cloning (Lee et al., 1987). The explanation of the two-hit hypothesis pioneered our understanding of the molecular basis of cancer (Knudson, 1971). A genetic predisposition to RB malignancy is inherited if a single nonfunctional copy of RB1 is inherited or present in the germline, a second hit in somatic tissue of the retina will then result in complete loss of function leading to uncontrolled cellular proliferation (Knudson, 1971). The RB1 gene exhibits allelic heterogeneity, with more than 1700 different pathogenic germline RB1 variants logged on the Leiden Open Variation Database (LOVD) (Fokkema et al., 2011). An array of different types of germline variants in RB1 can serve as an inherited predisposition, or “first hit” (Stenfelt et al., 2017). Sequence variants such as single nucleotide variants (SNV) leading to a loss-of function in the RB1 gene product as well as structural variants like whole gene deletions, copy number variation (CNV) (Xu et al., 2020) and rearrangements can render a germline copy of RB1 non-functional (Davies et al., 2021). Chromothripsis spanning the RB1 locus provides an additional mechanism of inactivating RB1 (McEvoy et al., 2014). With the emergence of sequencing technologies and increasing use thereof, two genetic anomalies with regards to RB was encountered that contradicted the RB1 two-hit hypothesis. Firstly, in some cases the presence of a pathogenic germline variant in RB1 is not sufficient to increase the risk to 4 develop RB or a retinoma neoplasm, as numerous families have a strong family history of a pathogenic variant in RB1 without the development or RB in successive generations. A nonfunctional RB1 copy confers genomic instability but additional genetic changes beyond RB1 may be required for the development of RB, the number of genetic modifications required is greatly debated (Dimaras et al., 2008; Corson & Gallie, 2007). Secondly, RB has been diagnosed in cases with a family history of the disease, indicative of a pathogenic RB1 variant according to the two-hit hypothesis, yet germline sequencing revealed two wild-type copies of RB1 (Rushlow et al., 2013), suggesting that germline mutations in other tumor predisposition genes beyond RB1 can act as putative drivers in RB malignancy (Akdeniz et al., 2019). 2.4. Locus heterogeneity observed in RB predisposition Genes other than RB1 have been linked to heritable RB aetiology. The notion of looking beyond RB1 as provided evidence to add to our knowledge of the genomic landscape of heritable RB. Table 1 summarizes cancer susceptibility genes that have been implicated in RB aetiology, paediatric cancers as well as retinal development pathways. Copy number analysis pipelines have been designed to detect unbalanced copy number variations, such as large deletions and duplications. In future the addition of copy number analysis should be performed in an aim to detect loss of function variants due to copy number variation in the cancer susceptibility genes mentioned in table 1 (Kooi et al., 2016), bridging the gap between sequence variants and larger structural variants. Changes at an epigenetic level have been proven to contribute to RB development, with promotor methylation of TSG beyond RB1 greatly influencing disease progression (Benavente & Dyer, 2015; McEvoy & Dyer, 2015). Non-coding RNAs and miRNAs have been functionally linked to RB tumorigenesis (Reis et al., 2012), creating microRNA (miRNA) signatures and multiomics techniques to be used in clinical settings as both diagnostic and prognostic markers (Delsin et al., 2019; Plousiou & Vannini, 2019). Germline loss of function mutations in the TSG p53 has been associated with multiple cancer predisposition syndromes and RB neoplasm development (Kato et al., 1996). Due to the important role p53 plays in regulating apoptosis in RB (Nork et al., 1997), loss of heterozygosity structural variants involving tumor protein 53 (p53) have been associated with RB (Kato et al., 1996). A study conducted 5 by Castéra, and colleagues discovered a significant contribution of E3 ubiquitin-protein ligase (MDM2) as a modifier gene in RB (Castéra et al., 2010) as RB progression is marked by MDM2 signaling pathway aberrations (Xu et at., 2009). MDM2 in conjunction with P53-Binding Protein (MDM4) regulates the p53 signaling pathway. MDM4 variants implicated in RB oncogenesis were found in a Brazilian cohort (de Oliveira Reis et al., 2012). The use of exome gene panels detected pathogenic variants in a heritable RB cohort from Istanbul with wild type RB1 linking RB oncogenesis with the retinoic acid pathway. Pathogenic variants were found in Dehydrogenase [quinone] 1 (NQO1), Fibroblast growth factor receptor 4 (FGFR4), C-Type Lectin Domain Containing 7A (CLEC7), Apolipoprotein C3 (APOC3), MutY DNA Glycosylase (MUTYH), UDP- Glucuronosyltransferase 1-A (UGT1A1), and RB disease segregated with a variant in Keratin 85 (KRT85) (Akdeniz et al., 2019). The downregulation of Cyclin Dependent Kinase Inhibitor 1A (CDK4I), also rereferred to as p16INK4a, is strongly associated with heritable RB but not sporadic RB (Indovina et al., 2009). Germline mutations in CDKN1A are linked to uncontrolled cellular proliferation, increasing the risk of developing a neoplasm (Carvalho et al., 2013). Pathogenic variants in oncogenes BHLH Transcription factor (MYCN) and Tuberous Sclerosis Complex 2 (TSC2) have also been proven to act as major drivers in RB oncogenesis (Francis et al., 2021). 6 Table 1: Genes implicated in the aetiology of retinoblastoma development Gene symbol Full gene name HGVS Reference transcript Literature reference Gene symbol Full gene name HGVS Reference transcript Literature reference RB1 Retinoblastoma 1 NM_000321.3 Knudson, 1971 MCCC2 Methylcrotonyl-CoA Carboxylase Subunit 2 NM_022132.4 Akdeniz et al., 2019 ACADS Short-chain acyl-CoA dehydrogenase NM_000017.3 Akdeniz et al., 2019 MDM2 E3 ubiquitin-protein ligase NM_002392.6 Epistolato et al., 2011 APC APC Regulator Of WNT Signalling Pathway NM_000038.6 Capasso et al., 2020 MDM4 P53-Binding Protein NM_001204172 de Oliveira Reis et al., 2012 APOC3 Apolipoprotein C3 NM_000040.2 Akdeniz et al., 2019 MLH1 MutL Homolog 1 NM_001258271.2 Capasso et al., 2020 BRCA1 BRCA1 DNA Repair associated NM_007294.4 Capasso et al., 2020 MSH2 MutS Homolog 2 NM_000251.3 Capasso et al., 2020 BRCA2 BRCA2 DNA Repair associated NM_000059.4 Capasso et al., 2020 MSH3 MutS Homolog 3 NM_002439.5 Capasso et al., 2020 C2 Complement 2 NM_000063.4 Akdeniz et al., 2019 MUTYHa MutY DNA Glycosylase NM_001128425.1 Akdeniz et al., 2019 CFB Complement Factor B NM_001710.5 Akdeniz et al., 2019 MYCN BHLH Transcription factor NM_005378.4 Francis et al., 2021 CLEC7Aa C-Type Lectin Domain Containing 7A NM_197947.2 Akdeniz et al., 2019 NQO1 Dehydrogenase [quinone] 1 NM_000903.2 Akdeniz et al., 2019 CDKN1A Cyclin Dependent Kinase Inhibitor 1A NM_000389.5 Carvalho et al., 2013 PAX6 Aniridia type II NM_001368894.2 Li et al., 2011 Meng et al., 2014 CREBBP CREB Binding Protein NM_001079846.1 Kooi et al., 2016 PTCH1 Patched 1 NM_001083606.3 Capasso et al., 2020 CX3CR1 C-X3-C Motif Chemokine receptor 1 NM_001171174.1 Akdeniz et al., 2019 p16INK4A Cyclin-dependent kinase inhibitor 2A (CDKN2A) NM_000077.5 Indovina et al., 2009 DSPPb Dentin Sialophosphoprotein NM_014208.3 Akdeniz et al., 2019 TP53 Tumor Protein P53 NM_000546.6 Epistolato et al., 2011 FGFR4 Fibroblast growth factor receptor 4 NM_002011.4 Akdeniz et al., 2019 RHAG Rh Associated Glycoprotein NM_000324.2 Akdeniz et al., 2019 FUT6 Fucosyltransferase 6 NM_000150.2 Akdeniz et al., 2019 RPGRIP1 RPGR Interacting Protein 1 NM_020366.3 Akdeniz et al., 2019 GBE1 Glycogen branching enzyme NM_000158.3 Akdeniz et al., 2019 SAMD11 Sterile Alpha Motif Domain Containing 11 NM_001385640.1 Kubo et al., 2021 GHRL Ghrelin and Obestatin prepropeptide NM_001134944.1 Akdeniz et al., 2019 SERPINA1 Alpha1AT NM_001002235.2 Akdeniz et al., 2019 GNPAT Glycerone-Phosphate O- Acyltransferase NM_014236.3 Akdeniz et al., 2019 SLC34A1 Solute Carrier Family 34 Member 1 NM_003052.4 Akdeniz et al., 2019 HBD Haemoglobin Delta Chain NM_000519.3 Akdeniz et al., 2019 SMAD5 SMAD Family Member 5 NM_001001419.3 Ueki & Reh, 2012 HFE Homeostatic Iron Regulator NM_000410.3 Akdeniz et al., 2019 TYR Tyrosinase NM_000372.4 Akdeniz et al., 2019 KRT85 Keratin 85 NM_002283.3 Akdeniz et al., 2019 TSC2 Tuberous Sclerosis Complex 2 NM_001114382.3 Francis et al., 2021 MBL2b Mannose Binding Lectin 2 NM_000242.2 Akdeniz et al., 2019 UGT1A1a UDP-Glucuronosyltransferase 1- A NM_000463.2 Akdeniz et al., 2019 7 3. Materials and Methods 3.1. Ethical considerations and consent This study was conducted as a sub-study under the parent study, Paediatric Cancer (PeCan) Project, Inherited cancer predisposing mutations in a cohort of childhood cancer patients. Ethical clearance was obtained from the University of the Witwatersrand Faculty of Health Sciences Human Research Ethics Committee (Medical) (HREC Medical) prior to the commencement of the research. Ethical clearance certificates for both the parent study, protocol number M180855, and this sub-study, protocol number M230782 are included in appendices A and B respectively. The parents or legal guardians of the participants provided written and/or verbal consent and assent from a subset of participants; copies of the informed consent documents employed in the study are included in appendix F. 3.2. Clinical presentation and patient demographics Eight paediatric patients diagnosed with RB were included in this study from the PeCan project. All eight patients are of black South-African ancestry. No family history of RB was reported in any of the patients. Table 2 summarizes the clinical features and demographics of the eight patients. Table 2: Clinical presentation of RB patients Patient ID a Sex b Age of diagnosis Laterality of RB PC26 Female 1 Year 8 Months Unilateral RB PC43 Female 1 Year 6 Months Unilateral RB PC50 Female 4 Years Unilateral RB PC79 Male 4 Months Unilateral RB PC90 Female 3 Year 6 Months Unilateral RB PC104 Female 8 Months Bilateral RB PC120 Male 3 Months Bilateral RB PC123 Male 9 Months Unilateral RB a Patient IDs used to anonymize patient information. b Sex as assigned at birth 8 3.3. Whole exome sequencing 3.3.1. DNA sample preparation and sample quality control Genomic DNA was extracted and purified from peripheral blood samples prior to the commencement of this project using the salt-out method (Maurya et al., 2013). DNA quantification commenced with assessing the concentration of double-stranded genomic DNA using the Qubit TM High Sensitivity assay and the Qubit TM fluorometer (ThermoFisher Scientific, Waltham MA, USA). Concentration of all samples were normalised to 10 ng/µl. 3.3.2. Library generation Target amplification was performed using Ion AmpliSeq TM Exome RDY reagents, followed by partial digestion of amplicons using the Ion AmpliSeq TM FuPa reagent. Ligation of Ion P1 Adapters and Ion Xpress TM Barcodes was completed as per ThermoFisher Scientific manufacturer’s guidelines (ThermoFisher Scientific, Waltham MA, USA). Barcoded libraries were purified using Agencourt TM AMPure XP Reagents (Beckman Coulter, Brea, CA, USA) followed by library amplification. Amplified libraries were purified in two rounds, again using Agencourt TM AMPure XP Reagents (Beckman Coulter, Brea, CA, USA). Libraries were quantified using the 4200 TapeStation TM (Agilent technologies, Santa Clara, USA) in conjunction with the prescribed High Sensitivity D5000 ScreenTape assay. The concentration of 200- 400 base pair fragments were measured for the purposes of quality control and diluted accordingly to facilitate the combination of exome libraries. 3.3.3. Templating and sequencing Two Ion AmpliSeq TM Exome RDY libraries were combined on a single Ion 540 TM Chip, templated in the Ion 540 TM Kit – Chef (ThermoFisher Scientific, Waltham MA, USA) as planned on Torrent SuiteTM software. Sequencing was performed using the Ion Torrent GeneStudio TM S5 Sequencer (ThermoFisher Scientific, Waltham MA, USA). Sequencing output included variant call files (VCF), binary alignment maps (BAM), index files and quality control reports. 9 3.4. Variant interpretation VCF files produced were annotated with Ensembl variant effect predictor (VEP) tool (GRCh37 version 110) (McLaren et al., 2016) available online from Ensembl 110 Genome Browser (https://www.ensembl.org) (Martin et al., 2023). Note that the decision was made to align all exomes to the human reference genome build 19 (GRCh37/Hg19), for the sake of consistency throughout the bioinformatics workflow. Annotation outputs included variant location and variant type, i.e., single nucleotide variants (SNVs) such as missense or nonsense variants, insertions, or deletions (indels), frameshift, or splice variant (Hunt et al., 2022). Annotation output also included variant allele frequency as reported by two genomic databases, 1000 genomes project (The 1000 Genomes Project Consortium et al., 2015) and the genome Aggregation Database (gnomAD) (Chen et al., 2022). All the above-mentioned databases were accessed between August and September 2023. First line of variant filtering was to prioritize the genes that have previously been reported to be implicated in RB predisposition and paediatric cancer predisposition. These genes are summarized in Table 1. Variants with a minor allele frequency (MAF) above 5% were filtered out. Lastly, variants that were predicted to have little to no impact on protein structure and/ or function were filtered out. The in-silico tools used to quantify variant impact on protein structure and function included Sort Intolerable from Tolerable (SIFT) (Ng & Henikoff, 2003), Polymorphism Phenotyping version 2 (POLYPHEN-2) (Adzhubei et al., 2010). Combined Annotation-Dependent Depletion (CADD) scores were allocated to SNVs and small indels based on how deleterious that variant is predicted to be (Rentzsch et al., 2019). Both SIFT and POLYPHEN-2 take the variant position’s degree of evolutionary conservation into account as part of predicting the impact of the variant on a protein level. These in silico tools predict what structural and functional changes may occur in a protein due to amino acid substitution (de la Campa et al. 2017). Lastly, variants of interest were modelled on MutationTaster2021 to distinguish polymorphisms from mutations (Steinhaus et al., 2021). Coverage and the quality of variant calling was assessed for the shortlisted variants by viewing binary alignment maps (BAM) and their index files in an integrative genomics viewer (IGV) (Robinson et al., 2011), variants with 30x coverage or greater were considered to be true variant calls. Variants were classified according to The American College of Medical Genetics and Genomics and Association for Molecular Pathology (ACMG/AMP) guidelines (Richards et al., 2015). Previously reported variants were analysed based on ClinVar submissions (Landrum et al., 2018). https://www.ensembl.org/ 10 4. Results 4.1. Clinical presentation of patients The eight patients had a wide distribution of age of onset, ranging from as early as 3 months to as late as 4 years. Mean age of onset for RB cohort was 19 months with a standard deviation of 17.22 months. Both male (37.5%) and female (62.5%) patients were included in the cohort. The two patients (25%) with the earliest age of onset (3 months and 8 months respectively) were the only patients that presented with bilateral RB, while the remaining six patients (75%) presented with unilateral RB (onset age range ranging from 4 months to 48 months). 4.2. Variant annotation Whole exome sequencing (WES) of eight paediatric RB patient with a black South African ancestry collectively returned 353 655 annotated variants spanning the entire genome. Figure 1 depicts the distribution of variant consequences as observed in the 8 patient cohort. The largest number of variants were 122 011 intronic variants, accounting for 34.5%. These intronic variants were filtered out immediately. The second highest proportion of variants were 54 392 synonymous variants (15.38%), followed by 38 018 missense variants (10.75%). The 25 640 downstream variants made up 7.25% of the total number of variants. A total of 21 679 variants (6.13%) were detected in regulatory regions, and 18 143 variants (5.13%) in upstream regions. Collectively, non-coding transcripts, non-coding transcript exon variants and splice site polymorphisms contributed 73 772 variants (20.86%), grouped as other in Figure 1. The variants in coding regions were mainly synonymous and missense variants as depicted in Figure 2. A total of 4 538 novel variants were annotated. Figure 1: Distribution of annotated variants The majority of the sequence variants annotated were intronic variants, 122 011 intronic variants accounting for 34.5% of all variants, followed by 54 392 synonymous variants (15.38%). Thus approximately half (49.9%) of the annotated variants were filtered out at a very early stage of variant prioritization. 11 Figure 2: Distribution of variants in coding regions The vast majority of annotated variants in the coding region predicted to have an impact on protein structure and function were 38 018 missense variants 4.3. RB1 sequence variants All exonic variants in RB1 are tabulated in appendix C. Two benign RB1 sequence variants, NM_000321.3:c.1574C>G (NP_000312.2:p.Ala525Gly) and NM_000321.3:c.45_53del (NP_000312.2:p.Ala16_Ala18del) were detected in a single patient, PC43. The beforementioned in- frame deletion of three amino acids NM_000321.3:c.45_53del (NP_000312.2:p.Ala16_Ala18del) was shared in three of the eight patients (PC43, PC50 and PC123). A single missense variant NM_000321.3:c.1574C>G (NP_000312.2:p.Ala525Gly) was found in a single patient (PC26). 4.4. Variants of uncertain significance Variants of interest in each patient classified as variants of uncertain significance are summarized in Table 3, along with the ACMG/AMP codes applied to each variant. All of the variants are classified as variants of uncertain significance (VUS) because the ACMG/AMP codes applied could not categorize the variants as likely benign or likely pathogenic. Variants in blue are shared between individuals in the cohort and are discussed separately. An extended list of variants in cancer predisposing genes associated with RB, MAF < 5% are tabulated in appendix D. 12 A total of 12 frameshift variants predicted to have a detrimental impact on protein structure and protein folding were detected. All patients carried as least one frameshift variant. Two novel frameshift mutations were classified as VUS. A novel frameshift variant in PTCH1 NM_001385640.1:c.2386C>T (NP_001372569.1:p.Leu796Phe) resulting in a truncated protein was detected in patient 26 and novel frameshift mutation in PTCHD3 NM_001034842.4:c.152del, (NP_001030014.2:p.Pro51ArgfsTer69) detected in patient 120 diagnosed at a young age with bilateral tumor presentation. Two previously reported frameshift variants were detected in a single patient only. Frameshift variant in FUT6, rs150560931, caused by a single nucleotide insertion NM_000150.3:c.501_502insC (NP_000141.1:p.Tyr168LeufsTer230) and a frameshift variant in CREBBP, rs587783507, NM_001079846.1:c.5723del (NP_001073315.1:p.Pro1908HisfsTer30). The TSG MSH3 contained a novel inframe deletion, NM_002439.5:c.162_179del NP_002430.3:p.Ala57_Ala62del and a previously reported missense variant rs34168832 NM_002439.5:c.1817G>A (NP_002430.3:p.Ser606Asn) in two respective patients. The vast majority of variants were missense variants, two of which were novel. The novel missense variant were detected in APC NM_000038.6:c.1251T>G (NP_000029.2:p.Cys417Trp) and MBL2 NM_000242.3:c.34_35delinsGG (NP_000233.1:p.Leu12Gly). Three previously reported missense variants were predicted to be damaging by more than one in-silico prediction tool. DNA mismatch repair genes, MSH3, rs34168832, NM_002439.5:c.1817G>A (NP_002430.3:p.Ser606Asn) and MLH1 rs63751244 NM_001167617.3:c.1375G>A (NP_001161089.1:p.Glu459Lys). UGT1A1, NM_000463.3:c.22G>A (NP_000454.1:p.Gly8Arg) was also deemed a VUS. For patient PC43 diagnosed at 18 months, presenting with unilateral RB, a total of 14 variants in cancer predisposing genes were annotated, the greatest number of variants per patient. Twelve out of the fourteen variants were missense variants predicted to have a negligible impact on protein function. By factoring in Clinvar record for the existing missense variants, all were classified as likely benign based on codes PM2 and BP4. The frameshift variant SAMD11 NM_001385640.1:c.1890_1891del (NP_001372569.1:p.Lys631ArgfsTer71) variant in patient PC43 is shared between individuals in the cohort and deemed a VUS. Patient PC123 also carried the frameshift variant in SAMD11 NM_001385640.1:c.1890_1891del (NP_001372569.1:p.Lys631ArgfsTer71), along with a missense variant in HFE rs28934889. Table 4 contains the ACMG/AMP codes applied to shared variants. 13 Table 3: Subset of variants of interest as observed per patient Gene Variant HGVSc and Variant HGVSp rsID Consequence Impact a ACMG/AMP b PC 26 PTCH1 NM_000264.5:c.3913del, NP_000255.2:p.Asp1305ThrfsTer67 novel frameshift variant HIGH VUS (PM2, PP3, PM4) MLH1 NM_001167617.3:c.1375G>A, NP_001161089.1:p.Glu459Lys rs63751244 missense variant MODERATE VUS (PM2, PP3) UGT1A1 NM_000463.3:c.22G>A, NP_000454.1:p.Gly8Arg rs749552053 missense variant MODERATE VUS (PM2, PP3) PC 43 SAMD11 NM_001385640.1:c.1890_1891del, NP_001372569.1:p.Lys631ArgfsTer71 novel frameshift variant HIGH VUS (PM2, PP3,PM4 ) PC 50 APC NM_000038.6:c.1251T>G, NP_000029.2:p.Cys417Trp - missense variant MODERATE VUS (PM2, PP3) SMAD5 NM_001001419.3:c.1313-1_1313insC, NP_001001419.1:p.Asn438ThrfsTer11 - frameshift variant HIGH VUS (PM2, PP3, PM4) CREBBP NM_001079846.1:c.5723del, NP_001073315.1:p.Pro1908HisfsTer30 rs587783507 frameshift variant HIGH VUS (PM2, PP3, PM4) PC 79 SMAD5 NM_001001419.3:c.1313-1_1313insC, NP_001001419.1:p.Asn438ThrfsTer11 - frameshift variant HIGH VUS (PM2, PP3, PM4) MSH 3 NM_002439.5:c.1817G>A, NP_002430.3:p.Ser606Asn rs34168832 missense variant MODERATE VUS (PM2, PP3) PC 90 SAMD11 NM_001385640.1:c.1890_1891del, NP_001372569.1:p.Lys631ArgfsTer71 novel frameshift variant HIGH VUS (PM2, PP3, PM4) SMAD5 NM_001001419.3:c.1313-1_1313insC, NP_001001419.1:p.Asn438ThrfsTer11 - frameshift variant HIGH VUS (PM2, PP3, PM4) PC 104 SAMD11 NM_001385640.1:c.1890_1891del, NP_001372569.1:p.Lys631ArgfsTer71 novel frameshift variant HIGH VUS (PM2, PP3, PM4) PC 120 PTCHD3 NM_001034842.4:c.152del, NP_001030014.2:p.Pro51ArgfsTer69 - frameshift variant HIGH VUS (PM2, PP3, PM4) SMAD5 NM_001001419.3:c.1313-1_1313insC, NP_001001419.1:p.Asn438ThrfsTer11 - frameshift variant HIGH VUS (PM2, PP3, PM4) PC 123 SAMD11 NM_001385640.1:c.1890_1891del, NP_001372569.1:p.Lys631ArgfsTer71 novel frameshift variant HIGH VUS (PM2, PP3, PM4) HFE NM_139006.3:c.157G>A, NP_620575.1:p.Val53Met rs28934889 missense variant MODERATE VUS (PM2, PP3) a The impact of the variant of protein structure and function is a prediction based on scored collected from multiple in silico prediction tools, including SIFT, POLYPHEN-2 and CADD. b Codes in brackets applied according to ACMG/AMP guidelines to classify variant -: Previously reported variants without an rsID at the time of interpretation 14 4.5. Shared variants Several of the genes proposed as candidate genes in RB or paediatric cancer, as mentioned in Table 1 returned variants observed in multiple patients despite the low allele frequency in the general population. Table 4 depicts the shared variants, the frequency at which the mutation occurred in the eight patients in the study cohort and the allele frequency in the general population. Two frameshift variants, SAMD11 NM_001385640.1:c.1890_1891del (NP_001372569.1:p.Lys631ArgfsTer71) and SMAD5 NM_001001419.3:c.1313-1_1313insC (NP_001001419.1:p.Asn438ThrfsTer11) were found in half the patients. Coverage and quality of variant calling was evaluated by visualizing BAM files on an IGV. The IGV images of a select few variants are depicted in appendix E to demonstrate the BAM images utilized to assess variant calling quality. Table 4: Shared variants (SV) Gene Variant HGVSc notation and Variant HGVSp notation Variant frequency a MAF b rsID ACMG/AMP Classification c HFE NM_139006.3:c.157G>A NP_620575.1:p.Val53Met 2 (25%) 0.0002 rs28934889 VUS (PM2, PP3) KRT85 NM_002283.4:c.233G>A NP_002274.1:p.Arg78His 3 (37.5%) 0.0328 rs61630004 Likely benign (PM2, PP1, BP6) SAMD11 NM_001385640.1:c.1890_1891del NP_001372569.1:p.Lys631ArgfsTer71 4 (50%) - Novel variant VUS (PM2, PP3, PM4) SMAD5 NM_001001419.3:c.1313-1_1313insC NP_001001419.1:p.Asn438ThrfsTer11 4 (50%) - Novel variant VUS (PM2, PP3, PM4) a Variant frequency in cohort of eight RB patients b Minor allele frequency in general population from gnomAD c Codes in brackets applied according to ACMG/AMP guidelines to classify variant Variants in bold are novel variants predicted to have a deleterious impact on protein structure and function according to in-silico prediction tools, including SIFT, POLYPHEN-2 and CADD. The majority of variants classified as VUS were missense variants, however the variants predicted to have the largest impact on protein structure and function were the SAMD11 and SMAD5 frameshift variants shared between individuals. Both of the frameshift variants mentioned are novel variants occurring in 50% of the RB population of the study. 15 5. Discussion The germline genetic contribution to RB aetiology has evolved beyond RB1, but little to no investigation has probed into the genetic landscape of RB in South African patients of African ancestry. This study aimed to address this disparity and gap in genomic knowledge by testing a cohort of eight paediatric RB patients with a South African ancestry for germline sequence variants predisposing them to RB malignancy. WES sequencing data uncovered 22 variants in cancer predisposition genes with an uncertain significance and 4 538 novel variants distributed across the genome. The lack of functional and segregation data for the novel variants in cancer predisposition genes reduced them to a VUS classification. The correlation of the young age of onset with bilateral tumor presentation is characteristic of individuals carrying a germline mutation or “first hit” (Knudson, 1971). This correlation was observed in the cohort, patients PC104 and PC120 both presented with bilateral RB at a young age, exemplifying that the two hit hypothesis still holds true, the presence of a constitutional germline mutation in cancer susceptibility gene can be observed as bilateral RB presentation at a young age. No clear pathogenic or likely pathogenic sequence variant was observed in RB1 in patients PC104 or PC120. In approximately half of the patients, no exonic sequence variant impacting protein structure was observed in RB1, as presented in appendix 3. The absence of likely pathogenic or pathogenic sequence variants in RB1 describe the RB phenotype cements the notion that variants in cancer susceptibility genes beyond RB1 need to be investigated to determine the full germline mutational spectrum associated with heritable RB. Patient 104, who was diagnosed at 8 months with bilateral RB had a mere two sequence variants of interest in cancer predisposing genes, neither of which explain the RB phenotype. 5.1. RB1 variants The in-frame deletion NM_000321.3:c.45_53del NP_000312.2:p.Ala16_Ala18del present in three patients, has previously been described (rs572454921) and is predicted to be benign because it result in a negligible change in protein structure. One patient, PC 26 had a RB1 missense variant rs4151539 (NM_000321.3:c.1574C>G, NP_000312.2:p.Ala525Gly) previously described in the literature as probably pathogenic, in a Brazilian RB cohort that screened for RB1 mutations in both RB and retinoma patients (Barbosa et al., 2013). As the reporting of variants observed in sequencing data became good scientific practice, conflicting interpretations of this variant emerged. This variant is classified as likely benign. 16 5.2. Shared variants Variants shared between the patients in the RB cohort at a higher allele frequency than the allele frequency observed in the general population are of interest. The majority of these shared variants are found in genes associated with retinal metabolism and rod photoreceptor development (Gao et al., 2009). The variants observed are yet to be linked to RB with functional studies. The shared variants are depicted in blue and summarized in Table 3, with the ACMG/AMP codes applied. A VUS in the Homeostatic Iron Regulator (HFE) gene was detected in two individuals, previously identified as rs28934889, NM_139006.3:c.157G>A, NP_620575.1:p.Val53Met. Akdeniz and colleagues were the first to propose HFE as a candidate gene in RB (Akdeniz et al., 2019). The HFE gene is responsible for iron metabolism in the retina, optimal iron metabolism and regulation prevents oxidative damage due to iron overload (Gnana- Prakasam et al., 2010). The HFE complexed with other proteins regulate iron concentrations in the electron transport chain through the SMAD1/5/8 pathway (Gao et al., 2009). The KRT85 rs61630004 (NM_002283.4:c.233G>A NP_002274.1:p.Arg78His) missense mutation observed in three of the eight RB patients has been described in literature as a candidate gene in RB etiology (Akdeniz et al., 2019), suggesting the variant might segregate with disease, leading to the application of ACMG/AMP code PP1. A study conducted in Istanbul observed this variant in multiple individuals affected with RB, two of them in the same family, implying that the variant might segregate with disease. Due to the significantly higher frequency of variant in disease cohort compared to the general population ACMG/AMP code PM2 was applied. The prediction of a deleterious effect by an in-silico tool the variant was classified as pathogenic by Akdeniz and colleagues. The in-silico predictors used has since become outdated, improved versions of the same in-silico tools, SIFT (Ng & Henikoff, 2003) and POLYPHEN-2 (Adzhubei et al., 2010) now predict rs61630004 (NM_002283.4:c.233G>A NP_002274.1:p.Arg78His) to have inconsequential effects on protein structure and function. MutationTaster2021 (Steinhaus et al., 2021) classified this variant as a polymorphism as opposed to a damaging mutation. The application of ACMG/AMP codes PM2, PP1 and BP6 collectively lead to the current classification of KRT85 NM_002283.4:c.233G>A NP_002274.1:p.Arg78His as likely benign which is in accordance with Clinvar classification. The notably high frequency (50%) of a novel frameshift variant in SAMD11 NM_001385640.1:c.1890_1891del brings the impact of this SAMD11 on RB predisposition in question. The absence of this variant in population databases prompted the application of the ACMG/AMP code PM2. A truncated protein is produced, predicted to have a detrimental effect on protein function, as a result, ACMG/AMP codes PP3 and PM4 were 17 applied. Unfortunately, the genotype-phenotype correlation is difficult to establish. A recent functional study demonstrated that SAMD11 is involved in establishing rod photoreceptor identity in retinal cells during retinal cell differentiation and development by forming part of the rod-specific polycomb recessive complex (Kubo et al., 2021). Dysregulation of SAMD11 is one of the numerous genetic causes of X-linked recessive retinitis pigmentosa (Corton et al., 2016). A second novel frameshift mutation was observed in the SMAD5, a gene that similar to SAMD11 is expressed in retinal tissue. In vivo experiments provide functional proof that SMAD5 is activated to repair retinal cells in response to light damage. Identical to the SAMD11 variant discussed above, the novel nature of the SMAD5 variant prompted the application of the PM2 code. Furthermore, the PP3 code was applied because in-silico tools predicted this frameshift variant to be deleterious. SMAD5 is a component of the BMP-SMAD1/5/8 neuroprotective signaling pathway promote survival of retinal ganglion cells (Ueki & Reh, 2012). In half of the patients in the RB cohort, a germline NM_001001419.3:c.1313-1_1313insC variant in SMAD5 was observed, predicted to cause a lack of function. Future functional studies are needed to speak to whether the inability to repair retinal cells damaged by light can create genomic instability, increasing the likelihood of future mutations and clonal evolution. Both patients PC104 and PC120 presented with an early age of onset and bilateral tumours, suggestive of hereditary RB. The lack of sequence variants capable of rationalizing the RB phenotype shines a light on the limitation of analyzing sequence variants exclusively. 5.3. Variants of uncertain significance The frameshift mutation in CREB Binding Protein (CREBBP) NM_001079846.1:c.5723del (NP_001073315.1:p.Pro1908HisfsTer30), produces a truncated protein product. CREBBP has been established as a TSG in lymphoma (Zhang et al., 2017) with haploinsufficient function. Loss of function of CREBBP is the autosomal dominant genetic cause of Rubinstein-Taybi syndrome, a neurodevelopmental disorder associated with a predisposition to developing tumours of the nervous system (Roelfsema & Peters, 2007). Somatic CREBBP loss of function mutations and downregulation have been proven to be putative drivers in RB tumorigenesis (Kooi et al., 2016), however the impact of germline deleterious mutations in CREBBP on RB aetiology remains unknown, this variant has not been reported in combination with RB. PC 50, diagnosed with unilateral RB at the age of 4 years, presented with a germline variant in the Adenomatous polyposis coli gene (APC) NM_000038.6:c.1251T>G, NP_000029.2:p.Cys417Trp, TSG inactivated in a large proportion of colorectal cancers (Sparks et al., 1998), a substructure of APC called APC2 has been established as a TSG in RB (Singh et al., 2016; Beta et al., 2015).Wild-type APC antagonizes the WNT signaling pathway, to negatively regulate cell growth. Mutations in genes regulating the WNT signaling 18 pathway have been linked to vitreoretinopathies like retinopathy of prematurity (Drenser, 2016). Case studies have been reported where patients with regressed retinopathy developed RB after approximately 4 years, but due to the small sample size, a causative link between retinopathy and RB could not be established with adequate statistical power (Vasu et al., 2011). The late age of onset and unilateral RB presentation in PC 50 are indicative of sporadic RB, the absence of a driver mutation is reasonable. From the beforementioned study by Singh and colleagues, defective mismatch repair was implicated in RB (Singh et al., 2016) by increasing genomic instability. Two DNA mismatch repair genes, MLH1 and MSH3, observed in two patients, PC26 and PC79 respectively, returned variants deemed as variants of uncertain significance. Patient PC26 presented with a frameshift mutation in Patched gene 1 (PTCH1), predicted to be deleterious by in-silico prediction tools (ACMG/AMP code PP3 applied). PTCH1 is a TSG implicated in central nervous system cancers such as medulloblastoma (Skowron et al., 2021) and carcinoma due to dysregulation of the sonic hedgehog signaling pathway leading to inappropriate cellular proliferation (Doheny et al., 2020). The patient predicted to be genetically predisposed to RB malignancy due to early age of onset and bilateral tumours, patient (PC120) presented with a novel frameshift variant in PTCHD3 NM_001034842.4:c.152del, NP_001030014.2:p.Pro51ArgfsTer69 where a truncated protein is produced. When investigating the germline mutational spectrum of predisposing genes implicated in colorectal cancer, PTCHD3 was proposed as a candidate predisposition gene that may serve as a TSG (Smith et al., 2013). 5.4. Future prospects and limitations The majority of final variant classifications were VUS. In the case that functional data and segregation data becomes available for a variant classified as VUS, the variant will be revisited and reclassified in accordance with ACMG/AMP guidelines. The inability to find previously reported driver mutation in this cohort, especially in the patients with a young age of onset and bilateral RB, brings the utility of analyzing only sequence variants from WES data in question. This study had several limitations, mainly the failure of WES to detect large balanced structural anomalies and large deletions and duplications. The addition of copy number variation assays such as multiplex ligation-dependent probe amplification (MLPA) (Livide et al., 2012) or chromosomal microarrays (CMA) can complement WES sequencing data, producing a more comprehensive picture of all germline variants present in an individual. Epigenetic changes such as promotor methylation (McEvoy & Dyer, 2015) and non-coding RNA molecules (Plousiou & Vannini, 2019) that contribute to RB etiology went undetected when sequencing on a genomic level. 19 The underrepresentation of African populations in cancer genomics cohorts inevitably complicates variant interpretation, exemplified by the numerous novel variants found in this study. Cancer is an extremely heterogenous disease. The possibility of patients carrying a germline variant in genes not included in the curated list (Table 1) cannot be discounted. The study cohort included patients of both sexes, where the age of onset varied widely, and RB presentation was unilateral in some and bilateral in others. As the majority of RB cases are sporadic, it is reasonable to assume that some patients will not carry a predisposing constitutive variant. 6. Conclusion The utmost degree of sequence variant classification according to the ACMG/AMP guidelines was variant of unknown significance. The inability to link the RB phenotype of the testing cohort to a germline sequence variant in the genes previously associated with RB supports the beyond RB1 hypothesis. Although RB1 is likely to remain the most likely driver in RB etiology, a wider testing strategy including CNV will yield more informative results. Understanding the full mutational landscape of RB will aid in the construction of appropriate treatment plans for RB patients and genetic counselling for family members. Variant classification in understudied and underrepresented populations, like the black South African population is hampered by the lack of information available. The genomic data currently available is a far cry from being representative of a genetically diverse country like South Africa. This study adds to the ever- growing collection of variants found in the black South African population, a critical set piece to expanding the genomic research resources in South Africa. Competing interests The authors declare that they have no competing interests. Statement on data availability Data is not available in public domain at present. All variants will be made available on Clinvar https://www.ncbi.nlm.nih.gov/clinvar/ . Funding This study was funded by the Inherited cancer predisposing mutations in a cohort of childhood cancer patients study, principal investigator Dr Lindie Lamola. https://www.google.com/url?q=https://www.ncbi.nlm.nih.gov/clinvar/&sa=D&source=docs&ust=1701323194311506&usg=AOvVaw2JShiSz4ch-G3-uwaRmlMq 20 7. References Adzhubei, I. A., Schmidt, S., Peshkin, L., Ramensky, V. E., Gerasimova, A., Bork, P., Kondrashov, A. S., & Sunyaev, S. R. (2010). A method and server for predicting damaging missense mutations. 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Cancer Discovery, 7(3), 322–337. https://doi.org/10.1158/2159-8290.CD-16-1417 https://doi.org/10.1371/journal.pone.0038690 https://doi.org/10.4103/0301-4738.86322 https://doi.org/10.1016/j.cell.2009.03.051 https://doi.org/10.1080/13816810.2020.1799417 https://doi.org/10.1158/2159-8290.CD-16-1417 29 Appendices Appendix A: Human Research Ethics Committee (Medical), University of the Witwatersrand Ethics Certificate (M180855) Appendix B: Human Research Ethics Committee (Medical), University of the Witwatersrand Ethics Certificate (M230782) Appendix C: RB1 sequence variants in all patients Appendix D Extended list of variants of interest Appendix E BAM images to evaluate variant coverage Appendix F: Information, Consent and Assent forms Appendix G: Title Approval and Approved Research Protocol Appendix H: Plagiarism declaration and Turnitin report Appendix I: Journal Author Guidelines for Submissible format Appendix C: RB1 sequence variants in all patients Appendix 3 Table 5: RB1 Exonic variants Location Consequence HGVSc HGVSp Existing variant SIFT Polyphen-2 CADD_ PHRED AF a CLIN _SIG b PC26 13:48955458-48955458 m issense variant N M _000321.3:c.1574C>G N P_000312.2:p.Ala525G ly rs4151539 tolerated(0.07) probably dam aging(0.962) 24.7 0.0062 benign/likely benign, PC43 13:48878084-48878093 Infram e deletion N M _000321.3:c.45_53del N P_000312.2:p.Ala16_Ala18del rs572454921 13.38 0.0108 benign 13:48955516-48955516 synonym ous N M _000321.3:c.1632A>G N P_000312.2:p.Arg544% 3D rs143948310 12.48 0.0020 Benign, likely benign PC50 13:48878084-48878093 Infram e deletion N M _000321.3:c.45_53del N P_000312.2:p.Ala16_Ala18del rs572454921 13.38 0.0108 benign PC79 N o exonic variants PC90 N o exonic variants PC104 N o exonic variants PC120 N o exonic variants PC123 13:48878084-48878093 Infram e deletion N M _000321.3:c.45_53del N P_000312.2:p.Ala16_Ala18del rs572454921 13.38 0.0108 benign All exonic sequence variants in RB1 w ith a m inor allele frequency low er than 5 % . a Allele frequency as reported in gnom AD b Clinical significance as reported in Clinvar and ACM G/AM P variant classification codes 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 A B C D E F G H I J Appendix D Location Allele Consequence IMPACT SYMBOL Gene HGVSc HGVSp cDNA_position Existing_variation PC26 1:879381-879381 T missense_variant MODERATE SAMD11 148398 NM_001385640.1:c.2386C>T NP_001372569.1:p.Leu796Phe 2895 rs116362966 13:48955458-48955458 G missense_variant MODERATE RB1 5925 NM_000321.3:c.1574C>G NP_000312.2:p.Ala525Gly 1736 rs4151539 5:112173899-112173899 T missense_variant MODERATE APC 324 NM_000038.6:c.2608C>T NP_000029.2:p.Pro870Ser 2667 rs33974176 9:98209623-98209625 GT frameshift_variant HIGH PTCH1 5727 NM_000264.5:c.3913del NP_000255.2:p.Asp1305ThrfsTer67 4818-4820 - 12:52760957-52760957 T missense_variant MODERATE KRT85 3891 NM_002283.4:c.233G>A NP_002274.1:p.Arg78His 309 rs61630004 19:5831602-5831602 T missense_variant MODERATE FUT6 2528 NM_000150.3:c.977G>A NP_000141.1:p.Arg326Gln 1233 rs61739552 19:5832077-5832077 G frameshift_variant HIGH FUT6 2528 NM_000150.3:c.501_502insC NP_000141.1:p.Tyr168LeufsTer230 757-758 rs150560931 14:21811196-21811196 G missense_variant,splice_region_variantMODERATE RPGRIP1 57096 NM_001377523.1:c.1319A>G NP_001364452.1:p.Asp440Gly 1385 rs17103671 3:37083760-37083760 A missense_variant,splice_region_variantMODERATE MLH1 4292 NM_001167617.3:c.1375G>A NP_001161089.1:p.Glu459Lys 1921 rs63751244 2:234668955-234668955 A missense_variant MODERATE UGT1A1 54658 NM_000463.3:c.22G>A NP_000454.1:p.Gly8Arg 40 rs749552053 5:79950727-79950736 - inframe_deletion MODERATE MSH3 4437 NM_002439.5:c.195_203del NP_002430.3:p.Pro67_Pro69del 258-266 rs60484572 22:29130456-29130456 A missense_variant MODERATE CHEK2 11200 NM_001005735.2:c.254C>T NP_001005735.1:p.Pro85Leu 312 rs17883862 PC43 1:878271-878273 - frameshift_variant HIGH SAMD11 148398 NM_001385640.1:c.1890_1891delNP_001372569.1:p.Lys631ArgfsTer71 2399-2400 - 1:879481-879481 C missense_variant MODERATE SAMD11 148398 NM_001385640.1:c.2486G>C NP_001372569.1:p.Gly829Ala 2995 rs113383096 13:48878084-48878093 - inframe_deletion MODERATE RB1 5925 NM_000321.3:c.45_53del NP_000312.2:p.Ala16_Ala18del 199-207 rs572454921 16:3778363-3778363 T missense_variant MODERATE CREBBP 1387 NM_004380.3:c.6685G>A NP_004371.2:p.Gly2229Ser 7482 rs139688311 3:39307063-39307063 A missense_variant MODERATE CX3CR1 1524 NM_001171172.2:c.938C>T NP_001164643.1:p.Ala313Val 1140 rs137947370 4:88533730-88533730 C missense_variant MODERATE DSPP 1834 NM_014208.3:c.392T>C NP_055023.2:p.Ile131Thr 512 rs61731009 12:52760957-52760957 T missense_variant MODERATE KRT85 3891 NM_002283.4:c.233G>A NP_002274.1:p.Arg78His 309 rs61630004 10:54531361-54531362 CC missense_variant MODERATE MBL2 4153 NM_000242.3:c.34_35delinsGGNP_000233.1:p.Leu12Gly 51-52 - 14:21816432-21816432 A missense_variant MODERATE RPGRIP1 57096 NM_001377523.1:c.1697G>A NP_001364452.1:p.Gly566Glu 1763 rs34725281 16:2133726-2133726 T missense_variant MODERATE TSC2 7249 NM_000548.5:c.3914C>T NP_000539.2:p.Pro1305Leu 4024 rs45517320 13:32953529-32953529 T missense_variant MODERATE BRCA2 675 NM_000059.4:c.8830A>T NP_000050.3:p.Ile2944Phe 9029 rs4987047 3:37092025-37092025 T missense_variant MODERATE MLH1 4292 NM_001258271.2:c.1945C>T NP_001245200.1:p.His649Tyr 1975 rs2020873 2:234545330-234545330 T missense_variant MODERATE UGT1A10 54575 NM_019075.2:c.162G>T NP_061948.1:p.Glu54Asp 208 rs148859354 16:2097712-2097712 A missense_variant,splice_region_variantMODERATE NTHL1 4913 NM_002528.7:c.113C>T NP_002519.2:p.Ala38Val 124 rs202082304 PC50 1:879481-879481 C missense_variant MODERATE SAMD11 148398 NM_001385640.1:c.2486G>C NP_001372569.1:p.Gly829Ala 2995 rs113383096 13:48878084-48878093 - inframe_deletion MODERATE RB1 5925 NM_000321.3:c.45_53del NP_000312.2:p.Ala16_Ala18del 199-207 rs572454921 5:112154980-112154980 G missense_variant MODERATE APC 324 NM_000038.6:c.1251T>G NP_000029.2:p.Cys417Trp 1310 - 5:135513085-135513085 C frameshift_variant,splice_region_variant,intron_variantHIGH SMAD5 4090 NM_001001419.3:c.1313-1_1313insCNP_001001419.1:p.Asn438ThrfsTer11 1758-1759 - 12:52760957-52760957 T missense_variant MODERATE KRT85 3891 NM_002283.4:c.233G>A NP_002274.1:p.Arg78His 309 rs61630004 17:41243800-41243800 T missense_variant MODERATE BRCA1 672 NM_007294.4:c.3748G>A NP_009225.1:p.Glu1250Lys 3861 rs28897686 16:3779210-3779217 GGGGGG frameshift_variant HIGH CREBBP 1387 NM_001079846.1:c.5723del NP_001073315.1:p.Pro1908HisfsTer30 5921-5927 rs587783507 PC79 4:88534326-88534326 A missense_variant MODERATE DSPP 1834 NM_014208.3:c.988G>A NP_055023.2:p.Ala330Thr 1108 rs201942511 9:98231233-98231233 T missense_variant MODERATE PTCH1 5727 NM_001083606.3:c.1597G>A NP_001077075.1:p.Glu533Lys 2048 rs62637629 5:135513085-135513085 C frameshift_variant,splice_region_variant,intron_variantHIGH SMAD5 4090 NM_001001419.3:c.1313-1_1313insCNP_001001419.1:p.Asn438ThrfsTer11 1758-1759 - 12:52758810-52758810 T missense_variant MODERATE KRT85 3891 NM_002283.4:c.565G>A NP_002274.1:p.Asp189Asn 641 rs112554450 6:49587034-49587034 G missense_variant MODERATE RHAG 6005 NM_000324.3:c.199T>C NP_000315.2:p.Phe67Leu 226 rs116356543 2:47637246-47637246 G missense_variant MODERATE MSH2 4436 NM_000251.3:c.380A>G NP_000242.1:p.Asn127Ser 416 rs17217772 Danielle Beukman Appendix D: Extended list of variants of interest 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 A B C D E F G H I J 5:80057418-80057418 A missense_variant MODERATE MSH3 4437 NM_002439.5:c.1817G>A NP_002430.3:p.Ser606Asn 1893 rs34168832 PC90 1:878271-878273 - frameshift_variant HIGH SAMD11 148398 NM_001385640.1:c.1890_1891delNP_001372569.1:p.Lys631ArgfsTer71 2399-2400 - 7:138430019-138430019 A missense_variant MODERATE ATP6V0A4 50617 NM_020632.3:c.1327A>T NP_065683.2:p.Asn443Tyr 1610 rs542662856 4:88534140-88534140 T missense_variant MODERATE DSPP 1834 NM_014208.3:c.802G>T NP_055023.2:p.Gly268Trp 922 rs61738508 5:135513085-135513085 C frameshift_variant,splice_region_variant,intron_variantHIGH SMAD5 4090 NM_001001420.3:c.1313-1_1313insCNP_001001420.1:p.Asn438ThrfsTer11 1599-1600 - 19:5832203-5832203 A missense_variant MODERATE FUT6 2528 NM_000150.3:c.376C>T NP_000141.1:p.Arg126Trp 632 rs111589452 12:52758810-52758810 T missense_variant MODERATE KRT85 3891 NM_002283.4:c.565G>A NP_002274.1:p.Asp189Asn 641 rs112554450 17:41243502-41243502 A missense_variant MODERATE BRCA1 672 NM_007300.4:c.4046C>T NP_009231.2:p.Thr1349Met 4159 rs80357345 PC104 1:878271-878273 - frameshift_variant HIGH SAMD11 148398 NM_001385640.1:c.1890_1891delNP_001372569.1:p.Lys631ArgfsTer71 2399-2400 - 5:79950707-79950725 - inframe_deletion MODERATE MSH3 4437 NM_002439.5:c.162_179del NP_002430.3:p.Ala57_Ala62del 238-255 - PC120 5:112173899-112173899 T missense_variant MODERATE APC 324 NM_000038.6:c.2608C>T NP_000029.2:p.Pro870Ser 2667 rs33974176 4:88533730-88533730 C missense_variant MODERATE DSPP 1834 NM_014208.3:c.392T>C NP_055023.2:p.Ile131Thr 512 rs61731009 6:47846362-47846362 C missense_variant MODERATE PTCHD4 442213 NM_001013732.4:c.2218A>G NP_001013754.3:p.Thr740Ala 2393 rs113836634 10:27703027-27703028 - frameshift_variant HIGH PTCHD3 374308 NM_001034842.4:c.152del NP_001030014.2:p.Pro51ArgfsTer69 249 - 5:135513085-135513085 C frameshift_variant,splice_region_variant,intron_variantHIGH SMAD5 4090 NM_001001419.3:c.1313-1_1313insCNP_001001419.1:p.Asn438ThrfsTer11 1758-1759 - 12:69218490-69218490 T intron_variant MODIFIER MDM2 4193 NM_001145337.3:c.505+59G>T - - rs148010327 13:32953529-32953529 T missense_variant MODERATE BRCA2 675 NM_000059.4:c.8830A>T NP_000050.3:p.Ile2944Phe 9029 rs4987047 2:234545898-234545898 A missense_variant MODERATE UGT1A10 54575 NM_019075.2:c.730C>A NP_061948.1:p.Leu244Ile 776 rs28969685 16:2096377-2096377 C splice_polypyrimidine_tract_variant,intron_variantLOW NTHL1 4913 NM_001318193.2:c.116-10C>G - - rs3211970 PC123 1:865584-865584 A missense_variant MODERATE SAMD11 148398 NM_001385640.1:c.659G>A NP_001372569.1:p.Arg220Gln 1168 rs148711625 1:878271-878273 - frameshift_variant HIGH SAMD11 148398 NM_001385640.1:c.1890_1891delNP_001372569.1:p.Lys631ArgfsTer71 2399-2400 - 1:879381-879381 T missense_variant MODERATE SAMD11 148398 NM_001385640.1:c.2386C>T NP_001372569.1:p.Leu796Phe 2895 rs116362966 13:48878084-48878093 - inframe_deletion MODERATE RB1 5925 NM_000321.3:c.45_53del NP_000312.2:p.Ala16_Ala18del 199-207 rs572454921 6:36651889-36651889 T missense_variant MODERATE CDKN1A 1026 NM_000389.5:c.11C>T NP_000380.1:p.Pro4Leu 101 rs4986866 4:88533730-88533730 C missense_variant MODERATE DSPP 1834 NM_014208.3:c.392T>C NP_055023.2:p.Ile131Thr 512 rs61731009 5:176524540-176524540 T missense_variant MODERATE FGFR4 2264 NM_001291980.2:c.2068C>T NP_001278909.1:p.Arg690Cys 2240 rs148143006 6:26091149-26091149 A missense_variant MODERATE HFE 3077 NM_139006.3:c.157G>A NP_620575.1:p.Val53Met 169 rs28934889 6:26091149-26091149 A missense_variant MODERATE HFE 3077 NM_139009.3:c.88G>A NP_620578.1:p.Val30Met 100 rs28934889 9:37006494-37006494 T missense_variant MODERATE PAX5 5079 NM_001280552.2:c.451G>A NP_001267481.1:p.Val151Ile 688 rs115889954 14:94847262-94847262 A missense_variant MODERATE SERPINA1 5265 NM_001127700.2:c.863A>T NP_001121172.1:p.Glu288Val 1083 rs17580 16:2134418-2134418 A missense_variant MODERATE TSC2 7249 NM_001114382.3:c.4126G>A NP_001107854.1:p.Gly1376Arg 4236 rs45466399 Appendix E BAM images to evaluate variant coverage RB1 Missense variant (Likely benign) NM_000321.3:c.1574C>G NP_000312.2:p.Ala525Gly RB1 Inframe deletion (Benign) NM_000321.3:c.45_53del NP_000312.2:p.Ala16_Ala18del SMAD5 insertion Frame shift variant (VUS) NM_001001419.3:c.1313-1_1313insC NP_001001419.1:p.Asn438ThrfsTer11 SAMD11 deletion Frame shift variant (VUS) NM_001385640.1:c.1890_1891del NP_001372569.1:p.Lys631ArgfsTer71 KRT85 Missense variant (Likely benign) NM_002283.4:c.233G>A NP_002274.1:p.Arg78His PTCHD3 deletion Frame shift variant (VUS) NM_001034842.4:c.152del NP_001030014.2:p.Pro51ArgfsTer69 CREBBP deletion Frameshift variant (VUS) NM_001079846.1:c.5723del NP_001073315.1:p.Pro1908HisfsTer30 MSH3 Missense variant (VUS) NM_002439.5:c.1817G>A NP_002430.3:p.Ser606Asn Appendix F: Information, Consent and Assent forms Appendix G: Title Approval and Approved Research Protocol Private Bag 3 Wits, 2050 Fax: 027117172119 Tel: 02711 7172076 Reference: Mrs Sandra Benn E-mail: sandra.benn@wits.ac.za 20 July 2023 Ms D Beukman Person No: 719425 0C Montpark drive Magalies unit 5 Randburg 2195 South Africa PAG Dear Ms Danielle Beukman Master of Science in Medicine: Approval of Title We have pleasure in advising that your proposal entitled RB1 and beyond: Determining genetic causes of Retinoblastoma in South-African patients has been approved. Please note that any amendments to this title have to be endorsed by the Faculty's higher degrees committee and formally approved. Yours sincerely Mrs Sandra Benn Faculty Registrar Faculty of Health Sciences CANDIDATE’S SURNAME: BEUKMAN FIRST NAME/S: DANIELLE STUDENT NUMBER: 719425 CURRENT QUALIFICATIONS: BSc Hons Experimental Physiology TEL: N/A CELL: 0845834985 E-MAIL: beukmandanielle@gmail.com FAX: n/a DEGREE FOR WHICH PROTOCOL IS BEING SUBMITTED: MSc Med Genomic Medicine PART-TIME OR FULL-TIME: FULL TIME FIRST REGISTERED FOR THIS DEGREE: TERM : 1 YEAR: 2023 DEPARTMENT: HUMAN GENETICS TITLE OF PROPOSED RESEARCH: RB1 and beyond: Determining genetic causes of Retinoblastoma in South-African patients CANDIDATE’S SIGNATURE: DATE: 7 July 2023 SUPERVISOR 1 (NAME & SURNAME): Dr. Lindie Lamola 50 % Supervision SUPERVISOR’S QUALIFICATIONS PhD SUPERVISOR’S DEPARTMENT Human Genetics SUPERVISOR’S ADDRESS / TEL / E-MAIL: lindie.lamola@wits.ac.za SUPERVISOR 2 (NAME & SURNAME): Nkateko Mayevu 50 % Supervision SUPERVISOR’S QUALIFICATIONS: MSc SUPERVISOR’S ADDRESS / TEL / E-MAIL: nkateko.mayevu@wits.ac.za SYNOPSIS OF RESEARCH: Introduction: Retinoblastoma (RB) is the most common intraocular malignancy in pediatric patients. Up to 31