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 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: (4226/5000)

References: 80/80

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

prenatal screening.

3

Unstructured abstract

Abstract: (244/250)

Retinoblastoma is the prototype genetic cancer: in one or both eyes of young children, most

retinoblastoma are initiated by biallelic mutation of the retinoblastoma tumor suppressor gene, RB1 in a

developing retinal cell. All those with bilateral retinoblastoma have heritable cancer, although 95% have

not inherited the RB1 mutation. Non-heritable retinoblastoma is always unilateral, with 98% caused by

loss of both RB1 mutant alleles from the tumor, while 2% have normal RB1 in tumors initiated by

amplification of the MYCN oncogene. Good understanding of retinoblastoma genetics supports optimal

care for retinoblastoma children and their families. Retinoblastoma is the first cancer to officially

acknowledge the seminal role of genetics in cancer, by incorporating “H” into the 8th Edition of Cancer

Staging (2017): those who carry the RB1 cancer-predisposing gene are H1; those proven to NOT carry the

familial RB1 mutation are H0; we suggest H0* for those with <1% risk to carry familial RB1 mutation

due to undetectable mosaicsmmosaicism; and those at unknown risk are HX.

Loss of RB1 from a susceptible developing retinal cell initiates the benign precursor, retinoma.

Progressive genomic changes result in retinoblastoma, and cancer progression ensues with increasing

genomic disarray. Looking forward, novel therapies are anticipated from studies of retinoblastoma and

metastatic tumor cells and the second primary cancers that the carriers of RB1 mutations are at high risk

to develop. Here, we summarize the concepts of retinoblastoma genetics for ophthalmologists to assist in

the care of patients and their families.

4

4381/5000 words

104/80 references

INTRODUCTION

Retinoblastoma is the most common childhood intraocular malignancy that affects one or both eyes.1

Because of the strong links between clinical care and genetic causation,2 retinoblastoma is considered the

prototype of heritable cancers.3 Worldwide, about 8,000 children are newly diagnosed with

retinoblastoma every year (1/16,000)1,4 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

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

tumor (trilateral).5 Without timely and effective treatment, retinoblastoma may spread through the optic

nerve to the brain, or via blood, particularly to bone marrow, and result in death.

“How can 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 alleles of the RB1 tumor suppressor gene, and remains in the inner nuclear layer of the retina

(Figure 1), perhaps because it is unable to migrate to the outer retina and function normally.1,6,7 The cell

susceptible to become cancer is 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, who likely previously had an

undetected small tumor (retinoma) present from childhood, that later became active.8,9 The mean age at

5

presentation is 12 months in bilateral disease and 24 months in unilateral disease, in high-income

countries, but significant delay in low-income countries impacts negatively on the children.10

“What causes 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 copy of the

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

second copy of the RB1 gene is also damaged in a developing retinal cell.1 The RB1 gene resides on

chromosome 13q14 and encodes the retinoblastoma protein (pRB), an important regulator of cell division

cycle in most cell types, and the first tumor suppressor gene discovered.11 Normally, dephosphorylated

pRB represses expression of the E2F gene, thereby blocking cell division.12-14 To resume cell division,

cyclin-dependent kinases re-phosphorylate pRB, releasing expression of E2F.15 In many cell types, loss of

the 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, allowing

uncontrolled cell division to initiate cancer.7

“What causes retinoblastoma to be unilateral versus bilateral?”

In heritable retinoblastoma (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 cells that become cancer, usually in both eyes (Figure 2A, B). The most common Often The M2

event is loss of the normal RB1 allele and duplication of the mutant M1 allele, so 2in that two copies

of the mutant RB1 gene remain (LOH, loss of heterozygosity).16 Heritable retinoblastoma

encompasses 45% of all reported cases:.17-19 with either bilateral (80%), unilateral (15%) or trilateral (5%)

tumors.1 Germline retinoblastoma carries an increased risk of second primary cancers, most commonly

osteosarcoma, leiomyosarcoma and melanoma. Germline RB1 mutations carry the risk of second primary

cancers, most commonly leiomyosarcoma, osteosarcoma, and melanoma.20 These patients may benefit

from regular surveillance for such cancers for over their lifetime.

6

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.21

“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.22,23 The majority of RB1 mutations arise de novo, unique to a specific patient

or family (Figure 2B). However, some recurrent mutations are found in 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.24,25

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.26,27 Advanced

paternal age increases risk for retinoblastoma,28 suggesting that genomic 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).1

“So, only RB1 mutation causes retinoblastoma?”

There are two answers to this question: (i) Loss of both RB1 alleles only causes retinoma, a benign

precursor to retinoblastoma and other genes are modified to cause progression to cancer;9 and (ii) 2% of

unilateral retinoblastoma have normal RB1 and are caused by a different gene.

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

translucent white mass, reactive retinal pigment epithelial proliferation and calcific foci.8 Retinoma may

7

be found retrospectively after a child develops retinoblastoma (Figures 2C, 3). Pathology of retinoma

reveals non-proliferative fleurettes.9,29 Comparison of adjacent normal retina, retinoma and retinoblastoma

showed loss of both RB1 alleles and early genomic copy number changes in retinoma, that were amplified

further in the adjacent retinoblastoma.9 Many retinoblastomas have underlying elements of retinoma.

Retinoma can transform to retinoblastoma even after many years of stability, so they need lifelong

monitoring.30

(ii) 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).3,31 Other less common

genomic alterations in retinoblastoma tumors include differential expression of specific microRNAs32 and

recurrent single nucleotide variants/insertion-deletions in the genes BCOR and CREBBP.33 In comparison

to the genomic landscape of other cancers, retinoblastoma is one of the least mutated, and epigenetic

modification of gene expression may play an important role in retinoblastoma progression33-35

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

patients have RB1+/+MYCNA tumors, with in which the MYCN oncogene is amplified (28-121 DNA copies

instead of the normal 2 copies).21 These children are diagnosed at median age 4.5 months compared to 24

months for non-heritable unilateral RB-/- patients, and the tumors are histologically distinct with advanced

features at diagnosis.

“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. The earliest signs of retinoblastoma detectable by parents are leukocoria (white

pupil), either directly or in photographs (photo-leukocoria) and strabismus when the macula is involved

by tumor.{Balmer, 2007 #8320}. Facial features and various degrees of hypotony and mental retardation

in 13q deletion syndrome can lead to discovery of retinoblastoma.36,37 If we know to look because a

8

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.38

Next, tumor seeds spread out of the main tumor into the subretinal space, or the vitreous cavity. as

appearing as dust, spheres or tumor clouds.{Munier, 2014 #11111} Vitreous seeding is associated with

loss of the cadherin 13 gene (chromosome 16q), a genomic change that may induce poor cohesive forces

between tumor cells.39,40

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, sometimes showing central retinal artery occlusion on

pathology.38,41 Untreated, retinoblastoma spreads into the optic nerve and brain, or hematogenous spread

occurs through choroid, particularly to grow in bone marrow. Tumor extension through sclera can present

as orbital extension and proptosis.

“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 2). 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).42 Nonsense and

frame-shift germline mutations that lead to absent or truncated dysfunctional pRB, result in 90% bilateral

retinoblastoma (nearly complete penetrance, der DER = 2). Partially functional RB1 mutant alleles, show

variable penetrance and expressivity (Figure 2C) with fewer tumors and later onset43, and some carriers

9

never develop retinoblastoma (der DER = 1-1.5) . Some reduced penetrance mutations reduce RB1

protein expressioninclude: (i) mutations in exons 1 and 2,44 (ii) mutations near the 3’ end of the gene in

exons 24 to 27,45,46 (iii) splice and intronic mutations47 and (iv) missense mutations.48 Counter intuitively,

large deletions encompassing RB1 gene and MED1 gene also cause reduced expressivity/penetrance,

because RB1-/- cells cannot survive in the absence of MED4.49 In comparison, patients with large deletions

with one breakpoint in the RB1 gene typically present with bilateral disease.50

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

substitution51,52 (Figure 2D) and c.1981C>T (p.Arg661Trp) missense mutation (der DER >1 from

maternal inheritance, der DER <1 from paternal inheritance).53 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 mutant allele is maternally inherited there is sufficient

tumor suppressor activity to prevent retinoblastoma development in only 10% of carriers. However,

when the mutant RB1 allele is paternally transmitted, very little pRB is expressed, leading to

retinoblastoma in 68% of carriers.

“What are the treatments and what governs the choice?”

Germline status and laterality at presentation highly direct treatment choices. In non-germline unilateral

retinoblastoma, enucleation is the preferred definitive treatment modality if there is anticipated poor

visual outcome. If promising vision is anticipated, chemotherapy (systemic or intra-arterial) might be

utilized. In germline bilateral retinoblastoma, systemic chemotherapy followed by consolidation focal

therapy is preferred in symmetrical cases (8th edition TNMH cancer staging54 cT1b, cT2) (IIRC38 B, C, D

eyes). In asymmetrical cases, enucleation of the advanced eye and focal therapy for the less advanced eye

might be more appropriate.

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.54 Multiple staging systems have

10

predicted likelihood to salvage an eye without using radiation therapy, but published evidence is

confusing because significantly different staging versions have been used.1,54 The 2017 TNMH

classification is based on international consensus 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.54

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

outcomes (Figure 4Table 1): presence of a positive family history, bilateral or trilateral disease or high

sensitivity positive RB1 mutation testing, is stage H1; without these features before testing blood, HX;

and H0 for those relatives shown to not carry the proband’s specific RB1 mutation (Figure 2).54 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.1 Ancillary

therapies for specific indications include plaque radiotherapy and periocular chemotherapy. Intravitreal

chemotherapy for vitreous disease has recently dramatically improved safe eye salvage.55 For persons

carrying RB1 mutations, external beam radiation therapy is rarely indicated due to the high risk of

inducing later second cancers.1

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

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

11

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.56,57 Germline RB1 mutations carry the risk of

second primary cancers, most commonly leiomyosarcoma, osteosarcoma, and melanoma.57 Only after 30

years of being used on every child, the enormous impact of external beam irradiation was recognized:

more children with bilateral retinoblastoma who were treated with radiation, die of their second (third,

etc) cancer than of retinoblastoma.58,59

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

informed parents. Essentially, the doctors decide what treatment they “feel” is best, based on little

substantial evidence. There exists currently 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: eCancerCare encompassing the whole

medical record for a lifetime with retinoblastoma, including the genetic results, and viewable on line by

the family and patient,60 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?”

Untreated, retinoblastoma is lethal. If treated before metastasis occurs, cure is nearly 100%. Occasionally

metastatic retinoblastoma may be confused with a second cancer; molecular demonstration of the RB1

mutations of the intraocular retinoblastoma will confirm metastases.61 Globally, the chance for cure is

remote, and lack of knowledge of genetics results too often result in death of children who could have

been saved if they had surveillance and definitive treatment when tumors were small. We look forward to

the cancer genomic revolution focusing on metastatic retinoblastoma, leading to breakthrough drugs that

make retinoblastoma a zero death cancer. However, delayed diagnosis and treatment due to lack of

knowledge by ophthalmologists and parents, socioeconomic,56 and cultural factors are the major causes of

12

mortality. Asia and Africa have >70% mortality from retinoblastoma compared to <5% in developed

countries.62

“How can we test for retinoblastoma mutations?”

Genetic counseling is the psychosocial and educational process to help patients and families adapt to the

genetic risk, the genetic condition, and the process of informed decision-making.63,64 Especially when

genetic testing is not available or unaffordable, genetic counseling is very important. Concrete knowledge

of genetic test outcomes supports informed and precise genetic counseling. Genetic testing supports

precision medicine for those who need it (carry the RB1 mutant allele) and is more cost effective than

examining all at-risk family members.25,65

If the proband 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.24,66,67

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

first tests tumor DNA, and then look for the 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 2) for parents, siblings and

cousins.67 Quality of genetic results depends on quality of DNA. Fresh or frozen tumor samples are ideal,

but while formalin fixed paraffin embedded tumors generally produce highly degraded DNA. Whole

blood in EDTA or ACD anticoagulant provides high quality DNA.

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

appropriate technology depends on the clinical question being asked. Next-generation sequencing (NGS)

may be the most effective screening strategy to find an unknown de novo mutation in an affected proband,

13

and may detect lower level mosaic mutations.68,69 To screen family members for a known sequencing-

detectable RB1 mutation, targeted Sanger dideoxy-sequencing is still 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 methods suggest that NGS data can be interrogated for

copy number variants,68,70 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 known24 and can detect 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 identifying maternal cell contamination in prenatal diagnostic tests.

Epigenetic changes can also initiate retinoblastoma development.71 Hypermethylation of the RB1

promoter CpG island results in inhibition of RB1 gene transcription in 10-12% of retinoblastoma tumors,

commonly involving both alleles.25,72 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 chromosome, have been shown to cause constitutional RB1 promoter

hypermethylation.73,74

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

14

the RB1 transcript by reverse transcriptase PCR (RT-PCR).{{Zhang, 2008 #7502}{Zhang, 2008

#7502;Dehainault, 2007 #5872} RNA studies also clarify pathogenicity of intronic sequence

alterations. As costs decreases, whole genome sequencing 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.50 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.36

“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

(Figure 2, Table 2).1,67,75 If the proband’s mutation is not found in either parent, a risk for low level

mosaicism still exists. Offspring of any family member carrying the RB1 mutation can be tested during

pregnancy or immediately after birth. If the proband is mosaic for the RB1 mutation, parents and siblings

are not at risk, since mosaicism cannot be inherited. The children of a mosaic proband need to be tested as

early as possible, since if they do inherit the RB1 mutant allele, they will be a high risk of bilateral

disease. If they do not carry the mutation, they are at population risk for bilateral retinoblastoma.

15

“How does the RB1 status affect follow up of the child?”

If the child does non’t carry an RB1 germline mutation, no EUAs are required for the other eye with

follow-up only in clinic (Table 12). If the child carries a germline mutation or is mosaic for the RB1

mutation, the possibility of new tumors renders frequent EUAs essential to at least 3 years of age.76 The

germline status also predisposes to other second malignant neoplasms, requiring frequent annual

surveillance. The use of whole body MRI as a method for surveillance is controversial, as a high false

positive rate it might stillmay provoke unnecessary invasive procedures. Development of surveillance

programs beyond the pediatric age is a high priority for early detection of these often-lethal second RB1

induced cancers.59

“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, to involves

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

16 weeks gestation, by involves sampling 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

frequent with CVS,77 and is routinely assessed 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 wishdecide 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.77. Prenatal or

postnatal RB1 mutation testing will either show the baby to be “H0” (no family RB1 mutation) or “H1” (,

confirmed to carry the mutation). Early pre-term delivery of an H1 fetus baby achieves smaller tumors

16

with higher treatment success, eye preservation and visual outcome than delivery at full term (Figure

1x).43

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

choose to not do prenatal invasive testing. For 50% risk for retinoblastoma, it is important that the

pregnancy does not go past 40 weeks (Figure X25).43,67

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

In some countries, pre-implantation genetic diagnosis (PGD) with in vitro fertilization is an option.78

Conceptions are tested for the familial mutation at an early stage of embryonal development (typically at

the 8 cell stage). Those that do not carry the RB1 mutation are implanted. The procedure is costly, ranging

from $10,000-$15,000 CAD per cycle; 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.78

“Are these tests available worldwide?”

High sensitivity RB1 mutation testing is established in core labs mainly in high-income countries.79 In

low- and middle-income countries, genetic counseling as a specialty does not exist, so many families with

retinoblastoma children do not have the opportunity to understand the benefits of genetic testing and

counseling in retinoblastoma treatment and follow-up.1 In many places, existing health 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 and ,

increasing economic burden on affected families. As an example, Tthe Chinese government’s new policy

to allow families to have one additional more children will make the need to for genetic testing and

counseling even more iimportant. A novel international collaboration between the companies Impact

17

Genetics in Canada and Geneseeq in China is optimizing expertise to achieve high quality RB1 testing for

families.

In Egypt genetic testing for retinoblastoma is not available and genetic counseling is the only way to

address the issues.80 Ophthalmologists with insufficient training in retinoblastoma genetics are burdened

with the this task. Genetic counseling was found to increase knowledge gaps remaininged in the

translation of this knowledge into appropriate screening action.

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 genetic testing. Treatments

accommodate the risks of therapies, and surveillance protocols can achieve earlier diagnosis of

retinoblastoma and second cancers, leading to better outcomes. Knowledge of test results alleviates

psychological burden on families moving forward with their life choices for the affected child and future

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

18

Legends

Figure 1: Early awareness of risk for retinoblastoma optimizes therapy and outcomes. This child

was examined because his sibling (triplets) presented with retinoblastoma. His right eye appeared normal,

but optical coherence tomography (OCT) revealed two invisible tumors (* and ↓) (left images), which

were treated with laser therapy only (right images); OCT post laser shows coagulation of tumor, visible in

the retinal image.

Figure 2: Pedigrees illustrating inheritance patterns for RB1 mutations. A. Full penetrance and

expressivity for Null mutation: fathermother (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 exon

12; diseased eye ratio (derDER) = 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.Gly449XTer) RB1 missense1 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; derDER = 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) missense RB1 missense

mutation; the proband, II-1, later developed meningioma in the radiation field, and breast cancer; the

proband’s brother (II-3) and daughter (III-2) inherited the mutation and developed unilateral

retinoblastoma; der DER= 1.5. D. Parent of origin low penetrance51: c.607+1G>T RB1 splice mutation

that shows higher penetrance when inherited from father (ie II-1, III-1, III-3; derDER = 1, than from

mother (ie III-1, III-3; derDER = 0), likely due to increased expression from the maternal than the

paternal mutant RB1 allele;53 (overall derDER = 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

19

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.

Figure 3: Retinoma, the benign precursor of retinoblastoma (stable translucent retinal mass with

calcification and associate retinal pigment epithelial distrubance) was diagnosed in family C (figure 2)

only when his daughter (II1) was diagnosed with retinoblastoma. He never received treatment and was

alive and well at age 90 at last follow-up.

Figure 4: 8th edition TNMH Cancer Staging for intraocular retinoblastoma.54 A. Clinical Staging for

eyes with retinoblastoma, compared to IIRC38. B. HeritablityHeritability stage for persons at risk to carry

an RB1 germline mutation.

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

20

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.

Figure 5: Post-natal diagnosis of familial retinoblastoma to late to save good vision. Bilateral

inherited retinoblastoma in neonate with positive family history born full term (39 weeks) and examined a

5 days after birth: right eye stage cT2b (IIRC group C), left eye stage cT1b (IIRC group B) involving

both foveas; treatment with multiple periocular and systemic chemotherapies and 3 years of focal

therapies salvaged both eyes with legal blindness.

Fig. XX: Post-natal diagnosis of familial retinoblastoma too late to save good vision. Bilateral inherited

retinoblastoma in neonate with positive family history born full term (39 weeks) and examined a 5 days

after birth: right eye stage cT2b (IIRC group C), left eye stage cT1b (IIRC group B) involving both

foveas; treatment with multiple periocular and systemic chemotherapies and 3 years of focal therapies

salvaged both eyes with legal blindness.

Figure XX2: Early awareness of risk for retinoblastoma optimizes therapy and outcomes. This child was

examined because his sibling (triplets) presented with retinoblastoma. His right eye appeared normal, but

optical coherence tomography (OCT) revealed two invisible tumors (* and ↓) (left images), which were

treated with laser therapy only (right images); OCT post laser shows coagulation of tumor, visible in the

retinal image.

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

21

22

Table 1. Risks for probands and relatives to develop retinoblastoma and second cancers and clinical surveillance plans.75

2

Table X: 8th edition TNMH Cancer Staging for intraocular retinoblastoma: cT and Heritability. 54

2

References

1. Dimaras H, Corson TW, Cobrinik D, et al. Retinoblastoma. Nature Reviews Disease Primers. 2015:15021.

2. Knudson AG. Mutation and cancer: statistical study of retinoblastoma. Proceedings Of The National Academy Of Sciences Of The United States Of America. 1971;68(4):820-823.

3. Theriault BL, Dimaras H, Gallie BL, Corson TW. The genomic landscape of retinoblastoma: a review. Clin Experiment Ophthalmol. 2014;42(1):33-52.

4. Seregard S, Lundell G, Svedberg H, Kivela T. Incidence of retinoblastoma from 1958 to 1998 in Northern Europe: advantages of birth cohort analysis. Ophthalmology. 2004;111(6):1228-1232.

5. de Jong MC, Kors WA, de Graaf P, Castelijns JA, Kivela T, Moll AC. Trilateral retinoblastoma: a systematic review and meta-analysis. The lancet oncology. 2014;15(10):1157-1167.

6. Rootman DB, Gonzalez E, Mallipatna A, et al. Hand-held high-resolution spectral domain optical coherence tomography in retinoblastoma: clinical and morphologic considerations. Br J Ophthalmol. 2013;97(1):59-65.

7. Xu XL, Singh HP, Wang L, et al. Rb suppresses human cone-precursor-derived retinoblastoma tumours. Nature. 2014;514(7522):385-388.

8. Gallie BL, Ellsworth RM, Abramson DH, Phillips RA. Retinoma: spontaneous regression of retinoblastoma or benign manifestation of the mutation? Br J Cancer. 1982;45(4):513-521.

9. Dimaras H, Khetan V, Halliday W, et al. Loss of RB1 induces non-proliferative retinoma: increasing genomic instability correlates with progression to retinoblastoma. Hum Mol Genet. 2008;17(10):1363-1372.

10. Nyamori JM, Kimani K, Njuguna MW, Dimaras H. The incidence and distribution of retinoblastoma in Kenya. Br J Ophthalmol. 2012;96(1):141-143.

11. Friend SH, Bernards R, Rogelj S, et al. A human DNA segment with properties of the gene that predisposes to retinoblastoma and osteosarcoma. Nature. 1986;323(6089):643-646.

12. Nevins JR. The Rb/E2F pathway and cancer. Hum Mol Genet. 2001;10(7):699-703.13. Cobrinik D. Pocket proteins and cell cycle control. Oncogene. 2005;24(17):2796-2809.14. Sage J, Cleary ML. Genomics: The path to retinoblastoma. Nature. 2012;481(7381):269-270.15. Knudsen ES, Knudsen KE. Tailoring to RB: tumour suppressor status and therapeutic response.

Nat Rev Cancer. 2008;8(9):714-724.16. Cavenee WK, Dryja TP, Phillips RA, et al. Expression of recessive alleles by chromosomal

mechanisms in retinoblastoma. Nature. 1983;305(5937):779-784.17. MacCarthy A, Birch JM, Draper GJ, et al. Retinoblastoma: treatment and survival in Great

Britain 1963 to 2002. Br J Ophthalmol. 2009;93(1):38-39.18. Moreno F, Sinaki B, Fandino A, Dussel V, Orellana L, Chantada G. A population-based study of

retinoblastoma incidence and survival in Argentine children. Pediatr Blood Cancer. 2014;61(9):1610-1615.

19. Wong JR, Tucker MA, Kleinerman RA, Devesa SS. Retinoblastoma incidence patterns in the US Surveillance, Epidemiology, and End Results program. JAMA ophthalmology. 2014;132(4):478-483.

20. MacCarthy A, Bayne AM, Brownbill PA, et al. Second and subsequent tumours among 1927 retinoblastoma patients diagnosed in Britain 1951-2004. Br J Cancer. 2013;108(12):2455-2463.

21. Rushlow DE, Mol BM, Kennett JY, et al. Characterisation of retinoblastomas without RB1 mutations: genomic, gene expression, and clinical studies. The lancet oncology. 2013;14(4):327-334.

22. Lohmann DR. RB1 gene mutations in retinoblastoma. Hum Mutat. 1999;14(4):283-288.23. McEvoy J, Nagahawatte P, Finkelstein D, et al. RB1 gene inactivation by chromothripsis in

human retinoblastoma. Oncotarget. 2014.

3

24. Rushlow D, Piovesan B, Zhang K, et al. Detection of mosaic RB1 mutations in families with retinoblastoma. Hum Mutat. 2009;30(5):842-851.

25. Richter S, Vandezande K, Chen N, et al. Sensitive and efficient detection of RB1 gene mutations enhances care for families with retinoblastoma. Am J Hum Genet. 2003;72(2):253-269.

26. Zhu XP, Dunn JM, Phillips RA, et al. Preferential germline mutation of the paternal allele in retinoblastoma. Nature. 1989;340(6231):312-313.

27. Dryja TP, Morrow JF, Rapaport JM. Quantification of the paternal allele bias for new germline mutations in the retinoblastoma gene. Hum Genet. 1997;100(3-4):446-449.

28. Toriello HV, Meck JM, Professional P, Guidelines C. Statement on guidance for genetic counseling in advanced paternal age. Genet Med. 2008;10(6):457-460.

29. Tso MOM, Fine BS, Zimmerman LE. The nature of retinoblastoma. II. Photoreceptor differentiation: an electron microscopic study. Am J Ophthalmol. 1970;69(3):350-359.

30. Eagle RC, Jr., Shields JA, Donoso L, Milner RS. Malignant transformation of spontaneously regressed retinoblastoma, retinoma/retinocytoma variant. Ophthalmology. 1989;96(9):1389-1395.

31. Corson TW, Gallie BL. One hit, two hits, three hits, more? Genomic changes in the development of retinoblastoma. Genes Chromosomes Cancer. 2007;46(7):617-634.

32. Huang JC, Babak T, Corson TW, et al. Using expression profiling data to identify human microRNA targets. Nat Methods. 2007;4(12):1045-1049.

33. Kooi IE, Mol BM, Massink MP, et al. Somatic genomic alterations in retinoblastoma beyond RB1 are rare and limited to copy number changes. Sci Rep. 2016;6:25264.

34. Zhang J, Benavente CA, McEvoy J, et al. A novel retinoblastoma therapy from genomic and epigenetic analyses. Nature. 2012;481(7381):329-334.

35. Temming P, Corson TW, Lohmann DR. Retinoblastoma tumorigenesis: genetic and epigenetic changes walk hand in hand. Future Oncol. 2012;8(5):525-528.

36. Baud O, Cormier-Daire V, Lyonnet S, Desjardins L, Turleau C, Doz F. Dysmorphic phenotype and neurological impairment in 22 retinoblastoma patients with constitutional cytogenetic 13q deletion. Clin Genet. 1999;55(6):478-482.

37. Bojinova RI, Schorderet DF, Addor MC, et al. Further delineation of the facial 13q14 deletion syndrome in 13 retinoblastoma patients. Ophthalmic Genet. 2001;22(1):11-18.

38. Murphree AL. Intraocular retinoblastoma: the case for a new group classification. Ophthalmology clinics of North America. 2005;18:41-53.

39. Gratias S, Rieder H, Ullmann R, et al. Allelic loss in a minimal region on chromosome 16q24 is associated with vitreous seeding of retinoblastoma. Cancer Res. 2007;67(1):408-416.

40. Gustmann S, Klein-Hitpass L, Stephan H, et al. Loss at chromosome arm 16q in retinoblastoma: Confirmation of the association with diffuse vitreous seeding and refinement of the recurrently deleted region. Genes, chromosomes & cancer. 2011;50(5):327-337.

41. Valverde K, Pandya J, Heon E, Goh TS, Gallie BL, Chan HS. Retinoblastoma with central retinal artery thrombosis that mimics extraocular disease. Med Pediatr Oncol. 2002;38(4):277-279.

42. Lohmann DR, Brandt B, Hopping W, Passarge E, Horsthemke B. Distinct RB1 gene mutations with low penetrance in hereditary retinoblastoma. Human Genetics. 1994;94:349-354.

43. Soliman SE, Dimaras H, Khetan V, et al. Prenatal versus Postnatal Screening for Familial Retinoblastoma. Ophthalmology. 2016;123(12):2610-2617.

44. Sanchez-Sanchez F, Ramirez-Castillejo C, Weekes DB, et al. Attenuation of disease phenotype through alternative translation initiation in low-penetrance retinoblastoma. Hum Mutat. 2007;28(2):159-167.

45. Bremner R, Du DC, Connolly-Wilson MJ, et al. Deletion of RB exons 24 and 25 causes low-penetrance retinoblastoma. Am J Hum Genet. 1997;61(3):556-570.

46. Mitter D, Rushlow D, Nowak I, Ansperger-Rescher B, Gallie BL, Lohmann DR. Identification of a mutation in exon 27 of the RB1 gene associated with incomplete penetrance retinoblastoma. Fam Cancer. 2009;8(1):55-58.

4

47. Zhang K, Nowak I, Rushlow D, Gallie BL, Lohmann DR. Patterns of missplicing caused by RB1 gene mutations in patients with retinoblastoma and association with phenotypic expression. Hum Mutat. 2008;29(4):475-484.

48. Scheffer H, Van Der Vlies P, Burton M, et al. Two novel germline mutations of the retinoblastoma gene (RB1) that show incomplete penetrance, one splice site and one missense. J Med Genet. 2000;37(7):E6.

49. Dehainault C, Garancher A, Castera L, et al. The survival gene MED4 explains low penetrance retinoblastoma in patients with large RB1 deletion. Hum Mol Genet. 2014;23(19):5243-5250.

50. Mitter D, Ullmann R, Muradyan A, et al. Genotype-phenotype correlations in patients with retinoblastoma and interstitial 13q deletions. European journal of human genetics : EJHG. 2011.

51. Klutz M, Brockmann D, Lohmann DR. A Parent-of-Origin Effect in Two Families with Retinoblastoma Is Associated with a Distinct Splice Mutation in the RB1 Gene. Am J Hum Genet. 2002;71(1):174-179.

52. Schuler A, Weber S, Neuhauser M, et al. Age at diagnosis of isolated unilateral retinoblastoma does not distinguish patients with and without a constitutional RB1 gene mutation but is influenced by a parent-of-origin effect. European Journal Of Cancer. 2005;41(5):735-740.

53. Eloy P, Dehainault C, Sefta M, et al. A Parent-of-Origin Effect Impacts the Phenotype in Low Penetrance Retinoblastoma Families Segregating the c.1981C>T/p.Arg661Trp Mutation of RB1. PLoS Genet. 2016;12(2):e1005888.

54. Mallipatna A, Gallie BL, Chévez-Barrios P, et al. Retinoblastoma. In: Amin MB, Edge SB, Greene FL, eds. AJCC Cancer Staging Manual. Vol 8th Edition. New York, NY: Springer; 2017:819-831.

55. Munier FL, Gaillard MC, Balmer A, et al. Intravitreal chemotherapy for vitreous disease in retinoblastoma revisited: from prohibition to conditional indications. Br J Ophthalmol. 2012;96(8):1078-1083.

56. Soliman SE, Dimaras H, Souka AA, Ashry MH, Gallie BL. Socioeconomic and psychological impact of treatment for unilateral intraocular retinoblastoma. Journal Francais D Ophtalmologie. 2015;38:550—558.

57. Soliman SE, Ulster A, E. H, Toi A, MacDonald H, Gallie BL. Psychosocial determinants in treatment decisions in familial retinoblastoma Ophthalmic Genetics. 2016;In Press.

58. Eng C, Li FP, Abramson DH, et al. Mortality from second tumors among long-term survivors of retinoblastoma. J Natl Cancer Inst. 1993;85(14):1121-1128.

59. Temming P, Arendt M, Viehmann A, et al. How Eye-Preserving Therapy Affects Long-Term Overall Survival in Heritable Retinoblastoma Survivors. Journal Of Clinical Oncology. 2016.

60. Chiu HH, Dimaras H, Downie R, Gallie B. Breaking down barriers to communicating complex retinoblastoma information: can graphics be the solution? Can J Ophthalmol. 2015;50(3):230-235.

61. Racher H, Soliman S, Argiropoulos B, et al. Molecular analysis distinguishes metastatic disease from second cancers in patients with retinoblastoma. Cancer Genet. 2016.

62. Chantada GL, Qaddoumi I, Canturk S, et al. Strategies to manage retinoblastoma in developing countries. Pediatric blood & cancer. 2011;56(3):341-348.

63. Uhlmann WR. Response to Robert G. Resta commentary (Unprepared, understaffed, and unplanned: thoughts on the practical implications of discovering new breast and ovarian cancer causing genes). J Genet Couns. 2009;18(6):524-526.

64. Shugar A. Teaching Genetic Counseling Skills: Incorporating a Genetic Counseling Adaptation Continuum Model to Address Psychosocial Complexity. J Genet Couns. 2016.

65. Noorani HZ, Khan HN, Gallie BL, Detsky AS. Cost comparison of molecular versus conventional screening of relatives at risk for retinoblastoma. Am J Hum Genet. 1996;59(2):301-307.

66. Astudillo PP, Chan HS, Heon E, Gallie BL. Late-diagnosis retinoblastoma with germline mosaicism in an 8-year-old. J AAPOS. 2014;18(5):500-502.

5

67. Canadian Retinoblastoma S. National Retinoblastoma Strategy Canadian Guidelines for Care: Strategie therapeutique du retinoblastome guide clinique canadien. Can J Ophthalmol. 2009;44 Suppl 2:S1-88.

68. Li WL, Buckley J, Sanchez-Lara PA, et al. A Rapid and Sensitive Next-Generation Sequencing Method to Detect RB1 Mutations Improves Care for Retinoblastoma Patients and Their Families. J Mol Diagn. 2016;18(4):480-493.

69. Chen Z, Moran K, Richards-Yutz J, et al. Enhanced sensitivity for detection of low-level germline mosaic RB1 mutations in sporadic retinoblastoma cases using deep semiconductor sequencing. Hum Mutat. 2014;35(3):384-391.

70. Devarajan B, Prakash L, Kannan TR, et al. Targeted next generation sequencing of RB1 gene for the molecular diagnosis of Retinoblastoma. BMC Cancer. 2015;15(1):320.

71. Ohtani-Fujita N, Fujita T, Aoike A, Osifchin NE, Robbins PD, Sakai T. CpG methylation inactivates the promoter activity of the human retinoblastoma tumor-suppressor gene. Oncogene. 1993;8(4):1063-1067.

72. Zeschnigk M, Lohmann D, Horsthemke B. A PCR test for the detection of hypermethylated alleles at the retinoblastoma locus. J Med Genet. 1999;36(10):793-794.

73. Jones C, Booth C, Rita D, et al. Bilateral retinoblastoma in a male patient with an X; 13 translocation: evidence for silencing of the RB1 gene by the spreading of X inactivation. Am J Hum Genet. 1997;60(6):1558-1562.

74. Quinonez-Silva G, Davalos-Salas M, Recillas-Targa F, Ostrosky-Wegman P, Aranda DA, Benitez-Bribiesca L. "Monoallelic germline methylation and sequence variant in the promoter of the RB1 gene: a possible constitutive epimutation in hereditary retinoblastoma". Clin Epigenetics. 2016;8:1.

75. Impact Genetics I. Retinoblastoma Risks and Surveillance Plans. 2016; http://impactgenetics.com/wp-content/uploads/2012/10/Retinoblastoma-risks-and-surveillance-plans.pdf.

76. Valenzuela A, Chan HSL, Heon E, Gallie BL. A Language for Retinoblastoma: Guidelines Through Standard Operating Procedures. In: Reynolds J, ed. Pediatric Retina. New York: Marcel Dekker Inc; 2010:205-234.

77. Akolekar R, Beta J, Picciarelli G, Ogilvie C, D'Antonio F. Procedure-related risk of miscarriage following amniocentesis and chorionic villus sampling: a systematic review and meta-analysis. Ultrasound Obstet Gynecol. 2015;45(1):16-26.

78. Xu K, Rosenwaks Z, Beaverson K, Cholst I, Veeck L, Abramson DH. Preimplantation genetic diagnosis for retinoblastoma: the first reported liveborn. Am J Ophthalmol. 2004;137(1):18-23.

79. One Retinoblastoma World. 2015; One Retinoblastoma World is a global network with the bold idea that all children with retinoblastoma can have equal opportunity to optimal care. We aim for global collaboration to deliver coordinated, evidence-based retinoblastoma care. Available at: http://1rbw.org/. Accessed August 31 2015.

80. Soliman SE, ElManhaly M, Dimaras H. Knowledge of genetics in familial retinoblastoma. Ophthalmic Genet. 2016:1-7.

6