Format of the review article:

- A word limit of 5,000 words;

- Less than 80 references;

- No strict limit to the number of tables and figures (8-10 recommended);

- An unstructured abstract of ≤ 250 words;

- The maximum number of authors: 6

Genetics and Molecular Diagnostics in

Retinoblastoma - An Update

Authors:

Sameh E. Soliman, MD,1-2 Hilary Racher, PhD,3 Chengyue Zhang, MD,4 Heather MacDonald,1 Brenda L.

Gallie, MD.1,5

Affiliations:

1Department of Ophthalmology and Vision Sciences, University of Toronto, Ontario, Canada

2Department of Ophthalmology, Faculty of Medicine, University of Alexandria, Alexandria, Egypt.

3Impact Genetics, Bowmanville, Ontario.

4Department of Ophthalmology, Beijing Children’s Hospital, Capital Medical University, Beijing, China.

5Departments of Ophthalmology, Molecular Genetics, and Medical Biophysics, University of Toronto,

Toronto, Canada.

Corresponding author:

Brenda L. Gallie: Hospital for Sick Children, 555 University Ave, Toronto, Ontario, Canada M5G 1X8.

Telephone: +1-416-294-9729

email: brenda@gallie.ca

2

Disclosures:

Both SS and HR contributed equally to this review as first co-first authors.

We confirm that this manuscript has not been and will not be submitted elsewhere for publication, and all

co-authors have read the final manuscript within their respective areas of expertise and participated

sufficiently in the review to take responsibility for it and accept its conclusions.

HR is a paid employee and BG is an unpaid medical advisor at Impact Genetics. No other authors have

any financial/conflicting interests to disclose.

This paper received no specific grant from any funding agency in the public, commercial or not-for-profit

sectors.

Word Count: (/5000)

Key Words: retinoblastoma, RB1 gene, bilateral, unilateral, DNA sequencing, genetic counselling,

prenatal screening.

3

Unstructured abstract

Abstract: (120/250)

Retinoblastoma is an intraocular malignancy that affects one or both eyes of young children, that is

initiated by biallelic mutation of the retinoblastoma gene (RB1) in a developing retinal cell. A good

understanding of retinoblastoma genetics supports optimal care for retinoblastoma children and their

families. …..highlight the new things….The goal of this article is to simplify the concepts of

retinoblastoma genetics for ophthalmologists to assist in the care of patients and their families.

4

4134/5000 words

94/80 references

table x cTNMH

table x risk assessment derived from impact

table or discussion for surveillance for eye and lifetime

figure: small tumors and OCT image vs large tumors without surveillance

figure: pedigree: F445 parent of origin and lp

figure: pedigree potentially siotas?

Figure: potentially update to second cancers for Ramsey?

INTRODUCTION

Retinoblastoma is the most common childhood intraocular malignancy that affects one or both eyes.

{Dimaras, 2015 #10881} Because of the strong links between clinical care and genetic causation,

{Knudson, 1971 #11106} retinoblastoma is considered the prototype of heritable cancers.{Theriault, 2014

#8591} Worldwide, about 8,000 children are newly diagnosed with retinoblastoma every year (1/16,000)

{Seregard, 2004 #10380;Dimaras, 2015 #10881} but most have no access to knowledge of the important

role genetics plays in many aspects of retinoblastoma: clinical presentation, choice of treatment

modalities and follow-up for both child and family. We now highlight the genetic etiology of

retinoblastoma in the context of individual children and families, lead by the questions commonly asked

by parents.

“What is retinoblastoma?”

Retinoblastoma is a cancer that arises because both copies of the RB1 gene that normally suppresses

retinoblastoma, is lost from a developing retinal cell in babies and young children. Retinoblastoma can

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affect one (unilateral) or both eyes (bilateral) and, in 5% of children, is associated with a midline brain

tumor (trilateral).{de Jong, 2014 #10885} Without timely and effective treatment, retinoblastoma may

spread through optic nerve to the brain, or via blood, particularly to bone marrow, which will result in

death.

“How can this cancer show up at such a young age?”

The cell of origin of retinoblastoma is most likely a developing cone photoreceptor precursor cell that has

lost both copies alleles of the RB1 tumor suppressor gene, and remains in the inner nuclear layer of the

retina, , perhaps because it is unable to migrate to the outer retina and function normally.{Dimaras, 2015

#10881;Rootman, 2013 #11096;Xu, 2014 #9924} The susceptible cell susceptible to become that

becomes cancer is only present in the retinas of young children, from before birth, up to around 7 years of

age. Rarely, retinoblastoma is first diagnosed in older persons, but who likely there was previously had an

undetected small tumor (retinoma) present from childhood, that later became active.{Gallie, 1982

#10343;Dimaras, 2008 #13250} The mean age at presentation is 12 months in bilateral disease and 24

months in unilateral disease, in high-income countries, but is significantly delayed in low-income

countries.{Nyamori, 2012 #8389}

“What causesd retinoblastoma?”

No one knows what really causes the genomic damage to the RB1 gene, but retinoblastoma arises at a

constant rate in all races irrespective of local environment. In nearly 50% of patients the first RB1 gene is

damaged in most, or all, normal cells of the patient. A retinal tumor develops when the second RB1 gene

is also damaged in a developing retinal cell.{Dimaras, 2015 #10881} The RB1 gene on chromosome

13q14 encodes the retinoblastoma protein (pRB), an important regulator of cell division cycle in most cell

types, and the first tumor suppressor gene discovered.{Friend, 1986 #10882} Normally, dephosphorylated

pRB represses expression of the E2F gene, thereby blocking cell division.{Nevins, 2001

#15292;Cobrinik, 2005 #15298;Sage, 2012 #7850} To resume cell division, cyclin-dependent kinases re-

phosphorylate pRB, releasing expression of E2F.{Knudsen, 2008 #15310} In many cell types, loss of the

6

RB1 gene is compensated by increased expression of other related proteins. However, in susceptible cells,

such as retinal cone cell precursors, compensatory mechanisms are insufficient, leading toallowing

uncontrolled cell division,, and initiating cancer is initiated.{Xu, 2014 #9924}

“What causes retinoblastoma to be unilateral versus bilateral?”

In heritable retinoblastoma (sometimes also called germline retinoblastoma), the first RB1 allele (M1) is

mutant in nearly all cells, including germline reproductive cells, while the second allele (M2) is mutated

in the retinal cell that becomes initiating cancer. Often the M2 event in the retinal cell is loss of the

normal RB1 allele and duplication of the mutant M1 allele, so 2 copies of the mutant RB1 gene remain

(LOH, loss of heterozygosity). Heritable retinoblastoma encompasses 45% of all reported cases.

{MacCarthy, 2009 #8367;Moreno, 2014 #9935;Wong, 2014 #15170} with either bilateral (80%),

unilateral (15%) or trilateral (5%) tumors.{Dimaras, 2015 #10881} Germline retinoblastoma carries an

increased risk of second primary cancers, most commonly osteosarcoma, fibrosarcoma leiomyosarcoma

and melanoma. These patients can may benefit from regular surveillance for such cancers for their

lifetime.

Of non-heritable retinoblastoma, 98% have both RB1 M1 and M2 events within a retinal cell. In the

remaining 2%, retinoblastoma is induced by somatic amplification of the MYCN oncogene, in the

presence of normal RB1 genes.{Rushlow, 2013 #11102}”

“What caused these mutations? Did I cause them?”

No one is to blame for the mutations causing retinoblastoma. Many environmental forces induce DNA

damage, including cosmic rays, X-rays, DNA viruses, UV irradiation and smoking. The DNA damage

may be point mutations, small and large deletions, promotor methylation shutting down RB1 expression

and rarely, chromothripsis.{Lohmann, 1999 #9272;McEvoy, 2014 #8499} The majority of RB1 mutations

arise de novo, unique to a specific patient or family. However, some recurrent mutations are found in

7

unrelated individuals, such as those that affect 11 sites CpG DNA sequence sites, which are hyper-

mutable and make up 22% of all RB1 mutations.{Rushlow, 2009 #10337;Richter, 2003 #11998}

When there is no family history of retinoblastoma, a de novo RB1 germline mutation may arise either

pre- or post-conception. Pre-conception mutagenesis of RB1 usually occurs during spermatogenesis,

perhaps because cell division (and opportunity for mutation) is very active during spermatogenesis, but

not during oogenesis.{Zhu, 1989 #6514;Dryja, 1997 #15586;Munier, 1998 #10955} Advanced paternal

age increases risk for retinoblastoma,{Toriello, 2008 #15506} suggesting that base substitution errors

may increase in aging men. The affected child carries the de novo RB1 mutation in every cell, typically

presenting with 4-5 tumors and bilateral retinoblastoma. In contrast, if mutagenesis occurs post-

conception, during embryogenesis, only a portion of cells will carry the RB1 mutation (ie. mosaicism). If

the mutation arises during retinal development, the child will have unilateral retinoblastoma.{Dimaras,

2015 #10881}

So, only RB1 mutation causes retinoblastoma?

There are two answers to this question: (i) RB1 mutation only causes a benign precursor to

retinoblastoma, retinoma, and other genes are modified to cause progression to cancer;{Dimaras, 2008

#13250} and (ii) 2% of retinoblastoma have normal RB1 and are caused by a different gene.

In addition to loss of RB1, specific alterations in copy number of other genes are common in RB1-/-

retinoblastoma. There are gains (4-10 copies) in oncogenes MDM4, KIF14 (1q32), MYCN (2p24), DEK

and E2F3 (6p22), and loss of the tumor suppressor gene CDH11 (16q22-24).{Corson, 2007

#9909;Theriault, 2014 #8591} Other less common genomic alterations in retinoblastoma tumors include

differential expression of specific microRNAs{Huang, 2007 #8613} and recurrent single nucleotide

variants/insertion-deletions in the genes BCOR and CREBBP.{Kooi, 2016 #14338} In comparison to the

genomic landscape of other cancers, retinoblastoma is one of the least mutated.{Kooi, 2016 #14338}”

8

There is a newly recognized form of retinoblastoma with normal RB1 genes. Two percent of unilateral

patients have RB1+/+MYCNA tumors, with the MYCN oncogene is amplified (28-121 instead of the normal

2 DNA copies).{Rushlow, 2013 #11102} These children are diagnosed at median age 4.5 months

compared to 24 months for non-heritable unilateral RB-/- patients, and the tumors are distinct

histologically, with advanced features at diagnosis.

Retinoma is a premalignant precursor to retinoblastoma with characteristic clinical features:

translucent white mass, reactive retinal pigment epithelial proliferation and calcific foci.{Gallie, 1982

#10343} Pathology of retinoma reveals fleurettes{Tso, 1970 #3456} that are not proliferative.{Dimaras,

2008 #13250} Comparison of adjacent normal retina, retinoma and retinoblastoma shows in retinoma loss

of both RB1 alleles and early genomic copy number changes, that are amplified further in the adjacent

retinoblastoma.{Dimaras, 2008 #13250} Many retinoblastoma have underlying elements of retinoma.

Retinoma can transform to retinoblastoma even after many years of stability.{Theodossiadis, 2005

#5578}

Could we have discovered retinoblastoma earlier?

The only way to find retinoblastoma tumor early is to examine the eye with specific expertise, which

we cannot do for every child. If we know to look because a relative had retinoblastoma, the smallest

visible tumors are round, white retinal lesions that obscure the underlying choroidal pattern.

Centrifugal growth results in small tumors being round; more extensive growth produces lobular

growth, likely related to genomic changes in single (clonal) cells, that provide a proliferative advantage.

{Murphree, 2005 #11984;Balmer, 2006 #8323} Next, tumor seeds spread out of the main tumor a result

of poor cohesive forces between tumor cells into the subretinal space, or the vitreous cavity as appearing

as dust, spheres or tumor clouds.{Munier, 2014 #11111} Advanced vitreous tumor seeds can migrate to

the anterior chamber producing a pseudo-hypopyon. Enlarging tumor can push the iris lens diaphragm

forward causing angle closure glaucoma. Advanced tumors may induce iris neovascularization. Rapid

necrosis of tumor can cause an aseptic orbital inflammatory reaction resembling orbital cellulitis,

9

sometimes showing central retinal artery occlusion.{Balmer, 2007 #8320;Balmer, 2006 #8323;Murphree,

2005 #11984} Untreated, retinoblastoma spreads into the optic nerve and brain, or hematogenous spread

occurs through choroid, particularly to grow in bone marrow. Direct tumor growth through the sclera can

present as orbital extension and proptosis.

The earliest signs of retinoblastoma detectable by parents are leukocorea (white pupil), either

directly or in photographs (photo-leukocorea) and strabismus when the macula is involvement by tumor.

{Balmer, 2007 #8320} In developing countries, buphthalmos and proptosis due to advanced and

extraocular disease respectively is common.{Canturk, 2010 #13461} Less common presentations include;

heterochromia irides, neovascular glaucoma, vitreous hemorrhage, hypopyon or aseptic orbital cellulitis.

{Balmer, 2007 #8320} Retinoblastoma (unilateral or bilateral) might be associated with a brain tumor in

the pineal, suprasellar or parasellar regions (Trilateral retinoblastoma){Popovic, 2007 #9156;Antoneli,

2007 #10877} with the median age of diagnsosis 17 months after retinoblastoma and before the age of 5

years. Retinoblastoma might present as 13q deletion syndrome, with facial features and various degrees

of hypotony and mental retardation.{Baud, 1999 #8118;Bojinova, 2001 #13205;Skrypnyk, 2004 #5276}

The main differential diagnosis includes Coats’ disease, persistent hyperplastic primary vitreous and

ocular toxicariasis.{Balmer, 2007 #8320}

Do all affected individuals with RB1 mutations develop retinoblastoma?

Depending on the exact RB1 mutation, most, but not all, carriers of an RB1 mutation will develop

retinoblastoma and other cancers throughout life.

Each offspring of a person carrying an RB1 mutant gene has 50% risk to inherit the RB1 mutant gene

[Figure # Pedigree A, B – full penetrance]. A measure of expressivity of a mutant retinoblastoma allele is

the disease-eye-ratio (DER) (number of eyes affected with tumor divided by the number of carriers of the

mutation).{Lohmann, 1994 #10954} Nonsense and frame-shift germline mutations, which lead to absent

or truncated dysfunctional pRB, result in 90% bilateral retinoblastoma (nearly complete penetrance, der =

2). Partially functional RB1 mutant alleles, show reduced penetrance and expressivity (Figure # C) with

10

fewer tumors and later onset{Soliman, 2016 #15159}, and some carriers never develop retinoblastoma

(der = 1-1.5) . Some reduced penetrance mutations reduce RB1 protein expression: (i) mutations in exons

1 and 2,{Sanchez-Sanchez, 2007 #6108} (ii) mutations near the 3’ end of the gene in exons 24 to 27,

{Bremner, 1997 #12040;Mitter, 2009 #7216} (iii) splice and intronic mutations{Zhang, 2003

#8986;Schubert, 1997 #4830;Lefevre, 2002 #4903} and (iv) missense mutations.{Scheffer, 2000

#15178;Cowell, 1998 #10958} Strangely, large deletions encompassing RB1 gene and MED1 gene also

cause reduced expressivity/penetrance, because RB1-/- cells cannot survive in the absence of MED4.

{Dehainault, 2014 #12140;Bunin, 1989 #4280} In comparison, patients with large deletions with one

breakpoint in the RB1 gene typically present with bilateral disease.{Mitter, 2011 #7339;Matsunaga, 1980

#357;Albrecht, 2005 #10898}

There are two specific RB1 mutations showing a parent-of-origin effect: intron 6 c.607+1G>T

substitution{Klutz, 2002 #8593;Schuler, 2005 #5551} [Figure # D: reduced penetrance family with parent

of origin….] and c.1981C>T (p.Arg661Trp) (der >1 from maternal inheritance, der <1 from paternal

inheritance).{Eloy, 2016 #12079} Both may be explained by at differential methylation of intron 2

CpG85, which skews RB1 expression in favor of the maternal allele.{Buiting, 2010 #7661;Kanber, 2009

#16381} When the allele is maternally inherited there is sufficient tumor suppressor activity to prevent

retinoblastoma development and 90% of carriers remain unaffected. However, when the p.Arg661Trp

allele is paternally transmitted, very little RB1 is expressed, leading retinoblastoma in 68% of carriers.

What are the treatments and what governs the choice?

Treatment and prognosis from retinoblastoma depend on the stage of disease at initial presentation.

Factors predictive of outcomes include size, location of tumor origin, extent of subretinal fluid, presence

of tumor seeds and the presence of high risk features on pathology.{Mallipatna, 2017 #14252} Multiple

staging systems have predicted likelihood to salvage an eye without using radiation therapy, but published

evidence is confusing because significantly different versions have emerged.{Dimaras, 2015

#10881;Mallipatna, 2017 #14252} The 2017 TNMH classification is based on international consensus

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and evidence from an international survey of 1728 eyes, and separates well initial clinical and

pathological features predictive of outcomes to save the eye from retinoblastoma, in retrospective

comparison to 5 previous eye staging systems.{Mallipatna, 2017 #14252} (Table X)

Retinoblastoma is the first cancer in which staging recognizes the impact of genetic status on

outcomes: presence of a positive family history, bilateral or trilateral disease or high sensitivity positive

RB1mutation testing, is stage H1; without these features bfore testing blood, HX; and H0 for those

relatives shown to not carry the proband’s specific RB1 mutation.{Mallipatna, 2017 #14252} We propose

H0* for patients tested and having neither M1 and M2 RB1 mutant alleles of the tumor detectable in

blood, and parents showing no evidence of the M1 allele of their offspring, but with remaining low risk

(<1%) of undetectable mosaicism. Offspring of H0* persons who do not show their family’s RB1

mutation are H0; if they test H1 for the inherited mutation they need intense surveillance for eye tumors

from birth.

Choice of treatment depends on the laterality of disease, tumor stage and genetic status. Focal therapy

only can control cT1a eyes, but visually threatening or large cT1b tumors and cT2 eyes need

chemotherapy (systemic or intra-arterial chemotherapy) to reduce the size of the tumor followed by

consolidation focal therapies (laser therapy or cryotherapy) as initial treatment. Enucleation of eyes with

advanced tumors in unilateral disease where the other eye is normal is a definitive cure.{Dimaras, 2015

#10881} Ancillary therapies for specific indications include plaque radiotherapy and periocular

chemotherapy. Intravitreal chemotherapy for vitreous disease has recently dramatically improved safe eye

salvage.{Munier, 2012 #8588;Munier, 2012 #8587} For persons carrying RB1 mutations, external beam

radiation therapy is rarely indicated due to the high risk of inducing later second cancers.{Dimaras, 2015

#10881}

Saving life is the priority of retinoblastoma treatment, followed by vision salvage; the least important

is eye salvage. The child’s job is to play and develop in a healthy life; the many procedures and their

12

complications that may span years for at best a 50% chance to save a blind eye with risk of tumor spread,

are not justified, especially when the other eye is normal.{Soliman, 2015 #10948;Soliman, 2016 #14269}

However, often missing from choices in the complex care of children with retinoblastoma are the

truly informed parents. Essentially, the doctors decide, based on very little evidence, what treatment they

“feel” is best. There exists no easy way to show the parents prospectively the true “costs” of each

treatment: the burden of invasive therapies and potential complications; the imposition of hours and days

in hospitals and feeling ill on the child, whose real job in those critical, irreplaceable years, is to play; the

true costs including time off work, uncertainties; and the burden of “false hope” in the absence of real

evidence. There are imminent solutions on the horizon, such as eCancerCare encompassing the whole

medical record for a lifetime with retinoblastoma, viewable on line by the family and patient, and the

burgeoning field of patient reported outcomes. These new attitudes and tools may empower in the future

good choices by parents for their child and family.

Is retinoblastoma lethal?

If untreated, retinoblastoma is lethal. If treated before metastasis occurs, cure is nearly 100%. Globally,

the chance for cure is even more remote, and lack of knowledge of genetics results too often result in

death of many children who could have been saved if they had surveillance and definitive treatment when

tumors were small. If metastasis occurs, the treatment becomes challenging and there is around 40%

chance of mortality. Delayed diagnosis and treatment due to lack of knowledge by ophthalmologists and

parents, socioeconomic{Soliman, 2015 #10948} and cultural factors are major causes of mortality. Asia

and Africa have the highest mortality, with >70% of affected children dying of retinoblastoma, compared

with <5% in developed countries.{Chantada, 2011 #13420;Canturk, 2010 #13461} Broad understanding

of retinoblastoma genetics and genetic counseling can contribute to reducing mortality from

retinoblastoma.

Germline retinoblastoma carries the risk of development of second primary cancers, most commonly

leiomyosarcoma, osteosarcoma, and melanoma.{MacCarthy, 2013 #11093} When the enormous impact

13

of external beam irradiation was recognized after 30 years of being used on every child, it was discovered

that more children with bilateral retinoblastoma (H1) were dying of their second (third, etc) cancer than of

retinoblastoma.{Eng, 1993 #10933}

Occasionally metastatic retinoblastoma may be confused with a second cancer; blue round cell tumors

on cytopathology may not differentiate from retinoblastoma, but molecular demonstration of the same

RB1 mutations as the intraocular retinoblastoma will confirm metastases.{Racher, 2016 #13990}

How can we test for retinoblastoma mutations?

If the patient is bilaterally affected, the probability of finding a germline mutation in the RB1 gene in

DNA extracted from blood is high (97% in a comprehensive RB1 laboratory). In 3% of bilateral

retinoblastoma patients, the predisposing RB1 mutation cannot be detected. In these instances,

identification of M1 and M2 RB1 mutations in DNA from tumor can assist in the identification of a

germline mutation, including low level mosaic mutations.{Astudillo, 2014 #10893;Rushlow, 2009

#10337;Canadian Retinoblastoma, 2009 #14251}

Similarly, to detect the 15% of unilateral patients carrying a germline mutation, the optimal strategy is

to first test tumor DNA, and then investigate for these mutations in blood. If the blood is not found to

carry one of the tumor RB1 mutations, risk of germline status is reduced to <1% (Table) for parents,

siblings and cousins.{Canadian Retinoblastoma, 2009 #14251}

Quality of genetic results depends on quality of DNA. Fresh or frozen tumor samples are ideal, but

formalin fixed paraffin embedded tumors generally produce highly degraded DNA. For blood genomic

whole blood in EDTA or ACD, provide high quality DNA.{Banfi, 2007 #15789}

The RB1 gene can be mutated in many ways, best identified by a series of techniques. Single

nucleotide variants (SNVs) and small insertions/deletions can be identified by DNA sequencing (Sanger

dideoxy-sequencing or next-generation sequencing (NGS)) methods.{Singh, 2016 #19381;Li, 2016

#19404;Chen, 2014 #19419} The most appropriate technology depends on the clinical question being

14

asked. NGS may be the most effective screening strategy to investigate for an unknown de novo mutation

in an affected proband, and may have a lower limit of detection (analytic sensitivity) for mosaic

mutations.{Chen, 2014 #14457} To screen family members for a known sequencing-detectable RB1

mutation, targeted Sanger sequencing is more cost and time effective.

Large RB1 deletions or duplications that span whole exons or multiple exons typically cannot be

detected by DNA sequencing. Multiplex ligation-dependent probe amplification (MLPA), quantitative

multiplex PCR (QM-PCR) or array comparative genomic hybridization (aCGH) are used to identify RB1

deletions and duplications, and other genomic copy number alterations, such as MYCN amplification.

New developments in bioinformatics analysis suggest that NGS data can be interrogated for copy number

variants,{Devarajan, 2015 #9654;Li, 2016 #14646} but sensitivity is not yet optimized.

Somatic mosaicism can arise in either the presenting patient or their parent. Allele-specific PCR (AS-

PCR) has excellent sensitivity when the RB1 mutation is known{Rushlow, 2009 #10337} and can detect

mutations as low as 1% mosaicism.

The second mutational event in 70% of retinoblastoma tumors is loss of heterozygosity (LOH), a

common event associated with loss of the normal allele in tumor from individuals with an inherited

cancer predisposition syndrome.{Cavenee, 1983 #9210} Polymorphic microsatellite markers distributed

throughout chromosome 13 can be used to detect a change from a heterozygous state in blood compared

to the homozygous state in a tumor with LOH. Microsatellite marker analysis is also important in identity

testing and in maternal cell contamination in prenatal diagnostic tests.

Epigenetic changes can also initiate retinoblastoma development.{Ohtani-Fujita, 1993 #2258}

Hypermethylation of the RB1 promoter CpG island results in inhibition of RB1 gene transcription in 10-

12% of retinoblastoma tumors, commonly involving both alleles.{Richter, 2003 #11998;Zeschnigk, 1999

#15496} This epigenetic gene silencing event primarily occurs in somatic cells, but heritable RB1

promoter mutations and translocations disrupting RB1 regulatory sites or translocations involving the X

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chromosome, have been shown to cause constitutional RB1 promoter hypermethylation.{Quinonez-Silva,

2016 #12111} (Jones et al 1997 PMID: 9199583)

Rarely, no RB1 mutation is identified in the coding, promoter or flanking intronic sequence in blood

from a bilateral patient. Deep intronic sequencing alterations that disrupt RB1 transcription by interfering

with correct splicing in patients with retinoblastoma can be detected by analysis of the RB1 transcript by

reverse-transcriptase PCR (RT-PCR).{Zhang, 2008 #7502;Dehainault, 2007 #5872} RNA studies also

clarify pathogenicity of intronic sequence alterations.{Zhang, 2008 #7502;Dehainault, 2007 #5872} As

NGS costs continue to decrease, whole genome sequencing (WGS) may become the method of choice to

uncover deep intronic changes.

Karyotype, fluorescent in situ hybridization (FISH) or array comparative genomic hybridization

(aCGH) of peripheral blood lymphocytes can be used to identify large deletions and rearrangements in

retinoblastoma patients, including patient’s suspected of 13q14 deletion syndrome.{Caselli, 2007

#15862;Mitter, 2011 #7339} In parents of 13q14 deletion patients, karyotype analysis can be used to

investigate for balanced translocations, which increases the risk for retinoblastoma in subsequent

generations.{Baud, 1999 #8118}

What is done after finding the RB1 mutation?

Family members at risk to also carry the identified RB1 mutation are offered testing on blood samples

(Table from impact…).{Canadian Retinoblastoma, 2009 #14251;Dimaras, 2015 #10881} If the mutation

is found in neither parent, a small risk for low level mosaicism still exists, leaving a low level risk for

siblings. Offspring of any family member carrying the RB1 mutation can be tested during pregnancy or

immediately after birth (see below). If the proband is mosaic for the RB1 mutation, parents and siblings of

the proband are not at risk, since mosaicism cannot be inherited. However, the children of a mosaic

proband needs to be tested as early as possible, as their risk to inherit the predisposing RB1 mutation is up

to 50%; if they do carry the mutation, they are at population risk for bilateral retinoblastoma.

16

Can we use the known mutation to test my future children?

Prenatal genetic testing is can be performed in the course of the pregnancy. Two early procedures are

available: i) chorionic villus sampling (CVS) performed between 11-14 weeks gestation, which involves

obtaining a sample of the placenta either trans-vaginally or trans-abdominally; and ii) amniocentesis after

16 weeks gestation, which involving amniotic fluid trans-abdominally. The procedure-associated risk of

miscarriage of CVS is 1%, while amniocentesis is 0.1-0.5%. Maternal cell contamination is more

frequently with CVS,{Akolekar, 2015 #9479} and is assessed for by the clinical molecular lab.

If the fetus does not carry the mutation, the pregnancy can proceed with no further intervention. If the

fetus carries the familial mutation, the parents have several choices. Some may decide to stop the

pregnancy, while others may know they wish to continue the pregnancy regardless of test results. If the

parents are concerned by the risk of miscarriage they can consider late amniocentesis between 30-34

weeks gestation when the major complication is early delivery rather than miscarriage.{Akolekar, 2015

#9479}. Prenatal or postnatal RB1 mutation testing will either show the baby to be “H0” (for the family

RB1 mutation) or “H1”, confirmed to carry the mutation. If the fetus has the familial RB1 mutation, early

pre-term delivery achieves smaller tumors and with higher treatment success, eye preservation and visual

outcome than delivery at full term.{Soliman, 2016 #15159}

In many countries, the option for prenatal genetic testing is not available, and some parents may

choose to not do prenatal invasive testing. If the risk for retinoblastoma in the fetus is 50% it is important

that the pregnancy does not go past 40 weeks.{Soliman, 2016 #15159;Canadian Retinoblastoma, 2009

#14251}

Can we plan our next pregnancy to passing on this RB1 mutation?

In some countries, preimplantation genetic diagnosis (PGD) with in vitro fertilization is an option.

{Dhanjal, 2007 #9216;Dommering, 2004 #10248;Xu, 2004 #9246;Girardet, 2003 #9219} Conceptions

are tested for the familial mutation at an early stage of development (typically 8 cells). Those that do not

carry the RB1 mutation are implanted. The procedure is costly, ranging from $10,000-$15,000 per cycle;

17

in some countries, there may be full or partial coverage of the costs. The full medical implications of PGD

are not yet understood; there is emerging evidence of a low risk for epigenetic changes in the conception

as a result of the procedure. It is recommended that typical prenatal testing be pursued during the course

of the pregnancy to confirm the results.{Dhanjal, 2007 #9216;Dommering, 2004 #10248;Girardet, 2003

#9219;Xu, 2004 #9246}

What is genetic counseling?

Genetic counseling is a psychosocial and educational process for patients and families to help them adapt

to the genetic risk, the genetic condition, and the process of informed decision-making.{Uhlmann, 2009

#15690;Shugar, 2016 #15715;Shugar, 2016 #15725} When genetic testing is not available or

unaffordable, genetic counseling is still very important.

Concrete knowledge of the genetic test outcomes supports informed and precise genetic counseling

and the specificity of the genetic test reduces the need for other possible scenarios to be discussed.

Genetic testing to support precision medicine for only those who need it (carry the RB1 mutant allele) is

more cost effective than examining all at-risk family members.{Noorani, 1996 #13637;Richter, 2003

#11998}

If we know the mutation prenatally, is there any treatment to prevent

retinoblastoma from occurring?

Today there is no prevention of retinoblastoma, only treatment when the tumors are very small, even

invisible. But ……..bg to write

Are these tests available worldwide?

High sensitivity RB1 mutation testing is established in core labs mainly in high-income countries.{, 2015

#10824} In low- and middle-income countries, genetic counseling as a specialty does not exist, so many

families with retinoblastoma children do not understand the benefits of genetic testing and counseling in

retinoblastoma treatment and follow-up.{Dimaras, 2015 #10881} In many places, existing health

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insurance does not cover costs of genetic testing. Given all these obstacles, there is limited application of

genetic testing and genetic counseling worldwide, overall health care costs are increased to deal with late

diagnoses, increasing economic burden on affected families. The Chinese government’s new policy to

allow families to have one more child will make the need to genetic testing and counseling even more

iimportant. A novel international collaboration between the companies Impact Genetics in Canada and

Geneseeq in China is optimizing expertise to achieve high quality RB1 testing for families.

In Egypt{Soliman, 2016 #14713} genetic testing for retinoblastoma is not available and genetic

counseling is the only way to address the issues. Ophthalmologists with insufficient training in

retinoblastoma genetics are burdened with the task. Genetic counseling was found to increase knowledge

gaps remained in translation of this knowledge into appropriate screening action.{Soliman, 2016 #14713}

Conclusions

Retinoblastoma genetics is a core element in care of retinoblastoma patients and their families. Action

based on knowledge of genetics in retinoblastoma improves outcomes for the eye and points to life and

treatment strategies to reduce early mortality and alleviate suffering. The whole family benefits

economically and in health, by precision in diagnosis of risk, based on testing, to establish who carries the

RB1 cancer-predisposing gene (H1), who does NOT carry the gene (H0), who has <1% risk to carry the

gene (H0*) and who is not tested so at unknown risk (HX). Knowledge of the test results alleviates the

psychological burden on the families moving forward with their life choices regarding the affected child

and future siblings and exposure to environmental carcinogens for the whole family.

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Legends

Figure ##: Pedigrees illustrating inheritance patterns for RB1 mutations. A. Full penetrance and

expressivity for Null mutation: mother (bilaterally enucleated) and 2/2 bilaterally affected offspring (I-1

unilateral enucleation vision 1.0 remaining eye; all H1 with 11 base pair deletion in RB1 exon 12;

diseased eye ratio (der) = 2. B. No family history, new Null mutation: triplets [insert ref to dimaras

NRDP] diagnosed with bilateral retinoblastoma at age 2.5 months due to c.1345G>T (p.Gly449Ter) RB1

mutation resulting in no pRB; parents showed no evidence of the mutation but were considered H0*

because there remains a <1% risk of mosaicism in either parent; older sib is negative for the mutation,

therefore, H0; der = 2. C. 100% penetrance and variable expressivity: grandfather (I-1) was diagnosed

with bilateral retinoma when his daughter (II-1) was diagnosed with bilateral retinoblastoma, due to

c.1960G>T (p.Val654Leu) RB1 missense mutation; the proband’s brother (II-3) and daughter (III-2)

inherited the mutation and developed unilateral retinoblastoma; der = 1.5. D. Parent-of-origin low

penetrance:[insert ref Klutz 2002] c.607+1G>T RB1 splice mutation that shows higher penetrance

when inherited from father (ie II-1, IV-1, IV-3; der = 1, than from mother (ie III-1, III-3, V-2; der = 0),

likely due to increased expression from the maternal than the paternal mutant RB1 allele;[insert ref Eloy

2016] (overall der = 0.7); IV-1§ had a small unilateral tumor but died at 11 years of age due to radiation

induced secondary malignancies; IV-5^ has not been tested, but developed thyroid cancer. H1 = carries

RB1 mutant allele; H0 = does NOT carry the familial RB1 mutant allele; H0* = <1% risk to carry familial

RB1 mutant allele; HX = unknown risk to carry familial RB1 mutant allele.

Insert refs after paper all finished….into the legend.

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Table X. Risks for probands and relatives to develop retinoblastoma and second cancers and clinical surveillance plans.No molecular testing

Table X: 8th edition TNMH Cancer Staging for intraocular retinoblastoma: cT and Heritability.