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

Chengyue Zhang, MD.

Hilary Racher, PhD

Heather MacDonald

Brenda L. Gallie.

2 Affiliations:

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

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

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

2 Impact Genetics, Bowmanville, Ontario.

Corresponding author:

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

publication, and all coauthors 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. No 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.

2

Unstructured abstract

Abstract: mmmmmmm

Key Words: retinoblastoma, RB1 gene,

3

INTRODUCTION [JEFFRY]

Retinoblastoma is the most common intraocular malignancy in childhood that might affect one or

both eyes.1 It is initiated by biallelic mutation of the retinoblastoma gene (RB1) in a single precursor

retinal cell. The constitutional RB1 mutation predisposes individuals to developing retinoblastoma that

forms after the somatic mutation.2,3 The incidence of retinoblastoma is constant at one case in 165,000-

1820,000 live births, translating to about 89,000 new cases per year worldwide. 1,3

There Asia and Africa have the highest mortality of >70about 40-70% of children with

retinoblastoma, in Asia and Africa, compared with <53-5% in developed countries.4,5 Delayed diagnosis

and treatment due to lack of retinoblastoma knowledge by ophthalmologists and pertaining to

retinoblastoma of parents, socioeconomic6 and cultural factors and ophthalmologists is one of theare

major causes leading to thebehind low eye salvage rate and high mortality in developing countries. So the

good understanding of retinoblastoma genetics and the importance of genetic counseling is a suitablethe

optimal way to address above issue in certain extent. In this review, we highlight the RB1 mutation

categories, advanced molecular diagnosis of retinoblastoma and genetic counseling.

Clinical presentation [Sameh]

Natural History

Retinoblastoma starts as a rounded white retinal mass that gradually increases in size. At first, equal

centrifugal growth of the tumor preserving the rounded or oval shape occurs followed by a period of

differential growth period leading toproducing the lobular or nipple growth patternstumor appearance.7,8

Tumor seeding occurs to the subretinal space or the vitreous cavity due to theas a result of poor cohesive

forces between tumor cells appearing as dust, spheres or tumor clouds.9, this can be into the subretinal

space or the vitreous cavity. In advanced tumors, the tumor seeds might migrate to the anterior chamber

4

producing a hypopyon like appearance, the enlarging tumor might push the iris lens diaphragm causing

angle closure glaucoma or rarely the rapid necrosis within the tumor can cause an aseptic orbital

inflammatory reaction resembling orbital cellulitis.7,8,10 If untreated, retinoblastoma can spread along the

optic nerve and along the visual pathway to the brain. Retinoblastoma can spread into the choroidal blood

vessels and hematogenous spread occurs. Direct tumor growth through the sclera can cause orbital

extension and proptosis. 11

Retinoma (premalignant variant) is transparent and associated with pigmentary changes due to

reactive retinal pigment epithelial growth and calcific foci. It is stable and does not grow over time.12 It

can transform to retinoblastoma even after many years of stability.13

Clinical Features

Leukocorea (white pupil) is main clinical presentation usually detected by parents either directly or in

photographs (photo-leukocorea). Strabismus due early macular involvement is the second most

common.10 In developing countries, buphthalmos and proptosis due to advanced and extraocular disease

respectively represents a higher percentage.5 Less common presentations include; heterochromia irides,

neovascular glaucoma, vitreous hemorrhage, hypopyon or aseptic orbital cellulitis.10 Retinoblastoma

(unilateral or bilateral) might be associated with a brain tumor in the pineal, suprasellar or parasellar

regions (Trilateral retinoblastoma)14,15 that starts early; with the median age of onset 17 months after

retinoblastoma is diagnosed and before the age of 5 years.{Popovic, 2007 #11607;Antoneli, 2007

#14202;de Jong, 2015 #14413} ItRetinoblastoma might present in a syndromic form (13q deletion

syndrome) associated with some facial features as high and broad forehead, thick and everted ear lobes,

short nose, prominent philtrum and thick everted lower lip, bulbous tip of the noseassociated with various

degrees of hypotonea and mental retardation.16-18 (Baud et al 1999 PMID: ; Bojinova et al 2001 PMID: ;

Skrypnyk and Bartsch 2004 PMID:) The main differential diagnosis includes Coats’ disease, persistent

hyperplastic primary vitreous and ocular toxicariasis.10

5

Trilateral: In approximately 5% of heritable cases, in addition to retinal tumors in one or both eyes, a

brain tumor (pineal, suprasellar or parasellar) will develop, a condition termed trilateral retinoblastoma

(de Jong et al 2015 PMID: 26374932). The onset of the brain tumor is relatively early, with the median

age of onset 17 months after retinoblastoma is diagnosed and before the age of 5 years (de Jong et al 2014

PMID: 26374932). The survival outcome for trilateral Rb patients has improved over the last 2 decades,

from very few to nearly half of all patients and is dependent on early detection and small tumor size (de

Jong et al 2014 PMID: 26374932). Improved survival is largely due to the use of high-dose chemotherapy

and autologous stem-cell rescue.

Grouping/staging

Treatment and prognosis depend on the stage of disease at initial presentation. The main factors

involved in grouping are size and site of the tumor, amount of subretinal fluid, size and site of tumor

seeds and the presence of high risk features.19 Multiple grouping systems for the intraocular

retinoblastoma existed with the international intraocular retinoblastoma classification (IIRC)7 being the

most reliable in the last decade despite confusing modifications.1 Recently, it has been replaced by the

TNMH classification.19 The main factors involved in grouping are size and site of the tumor, amount of

subretinal fluid, size and site of tumor seeds and the presence of high risk features. (Table X)

Retinoblastoma is the first cancer to be staged by genetics in addition to the clinical features due to the

high impact of genetic status on management. If there is a positive family history, bilateral disease or

documented positive RB1mutation testing, the disease is staged as H1. Otherwise it is considered as most

likely H0. A true H0 is considered with documented negative proband’s RB1 mutation status.19

-Pedigree defining H0 (*define a true H0 vs most likely H0), H1, HX

6

Treatments

Multiple treatments are now available and the choice depends on the laterality of disease and the

grouping of the tumor. Chemotherapy (systemic or intraarterial chemotherapy) to reduce the size of the

tumor followed by consolidation focal therapies (Laser therapy or cryotherapy) is the main stay of

treatment.1 Enucleation for eyes with advanced tumors or in unilateral disease where the other eye is

normal is more appropriate and definitive. Other therapies include; intravitreal chemotherapy for vitreous

disease, plaque radiotherapy or periocular chemotherapy. External beam radiation therapy has extremely

limited indications nowadays due to its extensive cancer risks and complications.1

Metastasis and Second Cancers

Germline retinoblastoma carry the risk of development of second primary cancers most commonly

osteosarcoma and fibrosarcoma. Sometimes it might be confused with metastatic retinoblastoma. Fine

needle aspiration cytopathology has minimal role in differentiation as both metastasis and second cancers

appear as blue round cell tumors. Genetic molecular analysis might help to differentiate.20…. (Hilary to

write details and choose appropriate site) –Cite Racher paper

Add differential diagnosis? NO, ELSEWHERE IN JOURNAL ISSUE; BUT ONE SENTENCE

ONLY….MERGE THE ABOVE HEADINGS INTO TWO PARAS…AT MOST.

Add retinoblastoma/retinoma? ONLY THE GENETICS OF IT

Inheritance pattern [Hilary]

Knudson two-hit hypothesis:

7

In most cases, retinoblastoma develops when both copies of the RB1 gene are inactivated. This

concept was first formulated in 1971, when Knudson used retinoblastoma as the prototypic cancer to

derive the two-hit hypothesis (Knudson, 1971).21 In heritable retinoblastoma, the first mutational event is

inherited via the germinal cells, while the second event occurs in the somatic cells. In nonheritable

retinoblastoma, both mutation events occur in the somatic cells. Heritable retinoblastoma encompasses

45% of all reported cases (MacCarthy et al 2009; Moreno et al 2014; Wong et al {risk of subse malig

neoplasms in long term hereditary rb surviv…}2014).22-24 The clinical presentation of heritable

retinoblastoma consists of 80% bilateral and 15-18% unilateral (cite).1 In non-heritable retinoblastoma

the majority (98%) of cases have somatic biallelic RB1 loss in the tumor, while the remaining 2% have no

mutation in either copy of RB1 but instead have somatic amplification of the MYCN oncogene. 25

Heritable Retinoblastoma and Penetrance

In heritable retinoblastoma, the each offspring of a each patient has a 50% risk of inheriting the RB1

pathogenic change. Whether the individual for whom inherited the RB1 mutation develops

retinoblastoma depends on the RB1 DNA alteration. Typically, nonsense and frame-shift germline

mutations, which lead to absence of RB1 expression or truncated dysfunctional RB1 protein, show nearly

complete (90%) penetrance. Often the second mutational event in the retinal cell is loss of the second

RB1 allele (LOH, loss of heterozygosity). In these families the presentation is typically unilateral,

multifocal or bilateral retinoblastoma. In a smaller subset of hereditary retinoblastoma, reduced

expressivity and reduced penetrance is observed (citations). In these families, when retinoblastoma

develops, it is often late onset and less severe, presenting as unilateral, unifocal (reduced expressivity)

and in some carrier family member retinoblastoma never develops (reduced penetrance). The types of

reported RB1 mutations reported that result in reduced expressivity or /penetrance arepenetrance are

diverse. Many consist of mutations whichmutations that reduced RB1 protein the expression. of the RB1

protein. Examples include, (1) mutations in exons 1 and 2 25,26 (2) mutations in exons 26 and

2726,27{Mitter, 2009 #18935;Mitter, 2009 #7347} (3) intronic mutations28,29 (Schubert et al 1997 PMID:

8

9341870; Lefevre et al 2002 PMID: 12011162 ; ) and (4) missense mutations (cite).30,31 In addition, large

deletions that encompassing the RB1 gene and the MED1 gene cause reduced expressivity/penetrance

(Dehainault et al 2014 PMID: 24858910; Bunin et al 1989 PMID: 2915374 ; ).32,33 Dehainault et al showed

that RB1 -/- cells cannot survive in the absence of MED4. This can explain why pPatients with 13q14

deletion syndrome more often have unilateral tumors only, in comparison to patients with gross deletions

with one breakpoint in the RB1 gene whom typically present with bilateral disease.34-36Rb (Mitter et al

2011 PMID: ; Matsunaga et al 1980 PMID: ; Baud et al 1999; Albrecht et al 2002 PMID: ) T. One way in

which the severity of risk can be evaluated is through the disease-eye-ratio (DER) (Lohmann et al 1994)

.calculated by taking the number of eyes affected with tumors divided by the total number of eyes of

carriers within the family. 37 The DER is calculated by taking the number of eyes affected divided by the

total number of eyes of carriers within the family.

In some instances of hereditable reduced expressivity/penetrance retinoblastoma, the parental origin

impacts whether or not an individual develops retinoblastoma and subsequently whether their carrier

offspring are at risk to develop retinoblastoma, a phenomenon termed the parent-of-origin effect (Klutz et

al 2002 PMID: 12016586; Schuler et al 2004 PMID: 15763650; Eloy et al 2016 PMID: 26925970).38-40 Eloy

A recent study by Eloy et al40 helped shed light onproposed a potential molecular mechanism to explain

the parent-of-origin effect. Using the c.1981C>T (p.Arg661Trp) reduced penetrance/expressivity

missense mutation, the researchers discovered that differential methylation of the intron 2 CpG85 skews

RB1 expression in favourfavor of the maternal allele. In other words, when the p.Arg661Trp allele is

maternally inherited there is sufficient tumor suppressor activity to prevent RB development and; 90.3%

of carriers of maternally inherited p.Arg661Trp remain unaffected. However, when the mutation allele is

paternally transmitted, very little RB1 is expressed, leading to haploinsufficiency and RB development in

67.5% of cases. A similar inheritance pattern was also reported for the intron 6 c.607+1G>T substitution

(Klutz et al 2002 PMID: 12016586).38

9

Trilateral: In approximately 5% of heritable cases, in addition to retinal tumors in one or both eyes, a

brain tumor (pineal, suprasellar or parasellar) will develop, a condition termed trilateral retinoblastoma

(de Jong et al 2015 PMID: 26374932). The onset of the brain tumor is relatively early, with the median

age of onset 17 months after retinoblastoma is diagnosed and before the age of 5 years (de Jong et al 2014

PMID: 26374932). The survival outcome for trilateral Rb patients has improved over the last 2 decades,

from very few to nearly half of all patients and is dependent on early detection and small tumor size (de

Jong et al 2014 PMID: 26374932). Improved survival is largely due to the use of high-dose chemotherapy

and autologous stem-cell rescue.

13q deletion syndrome

In patients with large interstitial 13q14 deletions that include the RB1 gene, variable clinical features

are present in addition to retinoblastoma, termed 13q14 deletion syndrome. Common facial features

includes high and broad forehead, thick and everted ear lobes, short nose, prominent philtrum and thick

everted lower lip, bulbous tip of the nose and mental retardation (Baud et al 1999 PMID: ; Bojinova et al

2001 PMID: ; Skrypnyk and Bartsch 2004 PMID: ). Patients with 13q14 deletion syndrome more often

have unilateral tumors only, in comparison to patients with gross deletions with one breakpoint in the RB1

gene whom typically present with bilateral Rb (Mitter et al 2011 PMID: ; Matsunaga et al 1980 PMID: ;

Baud et al 1999; Albrecht et al 2002 PMID: ).

?mechanism ?non-allelic homologous recombination.

Mosaicism

{FIGURE ON MOSAICISM}

RB1 gene [Hilary]

Function: The RB1 gene, located on 13q14, encodes the RB protein, which is an important cell cycle

regulator and the first tumor suppressor gene ever discovered (Friend et al 1986 PMID: ).41 After a cell

10

completes mitosis, the RB protein is dephosphorylated, permitting it to bind to the promoter region of the

E2F transcription factor gene, thereby repressing transcription and inhibiting the progression of the cell

cycle from G1 to S phase (Nevins et al 2001 PMID: ; Cobrinik 2005 PMID: ; Sage et al 2012 PMID: ).42-44

In order for the cell to enter S phase, cyclin-dependent kinases phosphorylate RB, which removes the

ability of RB to bind to the E2F gene promoter (Knudsen and Knudsen 2008 PMID: ).45 RB functions to

regulate proliferation in most cell types (Cobrinik 2005 PMID:).43 Often, loss of RB1 is compensated by

increased expression of its related proteins, however, in certain susceptible cells, such as the retinal cone

cell precursors, compensatory mechanisms are not sufficient and tumorigenesis is initiated (Xu et al 2014

– Nature – Rb suppresses human cone-precur PMID).46

-?A and B pockets

-Also describe the role in genomic instability (Demaris. Rushlow)

RB1 Mutations

Different ways in which RB1 can be disrupted: There are many ways in which the function of the RB

protein is impaired including point mutations, small and large deletions, promotor methylation and

chromothripsis (Lohmann 1999 PMID: ; McEvoy et al 2014 PMID: ).47,48 The majority of RB1

mutations are de novo, unique to a specific patient or family, however, there are some known recurrent

mutations found across many unrelated individuals. One subset of recurrent mutations involved CpGOne

subset of recurrent mutations involve 11 CpG sites, which make up ~22% of all RB1 mutations (Rushlow

et al 2009 PMID: 19280657).49 The high recurrence of nonsense mutations at these sites is due to the

hypermutabilty and subsequent deamination of 5-methylcytosine (Richter et al 2003).50

The origin of a de novo RB1 mutation can arise either pre- or post-conception. Most often, pre-

conception mutagenesis occurmutagenesis occurs during spermatogenesis. (Munier et al 1998 PMID:

9837842; Dryja et al 1997 PMID: 9272170).51,52.51,52 Furthermore Furthermore, advanced paternal age has

been shown to increase risk for retinoblastoma.53 This is thought tomight be due to the larger number of

11

cell divisions during spermatogenesis than oogenesis andor the increased rate for base substitution errors

in aging men compared to women. In cases of pre-conception mutagenesis, the proband carries the de

novo RB1 mutation in every cell within their body and typically presents with bilateral retinoblastoma. In

contrast, post-conception RB1 mutagenesis occurs during embryogenesis. Depending on the

embryological stage of development, a few or numerous tissues may be mosaic for the RB1 mutation. If

the mutational event occurs during retinal development, the presentation is often unilateral

retinoblastoma.1

Coding sequencing mutations

Promoter methylation

Hot-spot mutations – CpG transition

Non-coding/regulatory changes

?in genetic counselling?? Origin of new mutations

Xu et al. new mutations are on fathers chromosome

Older fathers, but not older mothers for RB50

Greta Bunin

MYCN

PROGRESSIVE OTHER GENOMIC CHANGES IN ADDITION TO RB1

Other genomic changes in addition to alterations in RB1 [Hilary]

DEK, KIF14, E2F3, CDH11

In a small subset (2%) of unilateral patients, no RB1 mutant is identified. Instead, striking

amplification (28-121 copies) of the MYCN oncogene is detected (Rushlow et al 2013 PMID: 23498719).25

12

Patients with RB1+/+ MYCNA are clinically distinct from RB-/- patients, showing much younger age at

diagnosis, distinct histological features and larger, more invasive tumors.

In addition to loss of RB1 or MYCN amplification, specific somatic copy number alterations

commonly occur in the progression of the retinoblastoma. Commonly seen are gains in 1q32, 2p24, 6p22

and losses at 13q and 16q22-24 (Corson and Gallie 2007 PMID: 17437278).2 These regions contain

important oncogenes (MDM4, KIF14, MYCN, DEK and E2F3) and tumor suppressor genes (CDH11),

thought to act as drivers promoting the growth of the cancer (Theriault et al 2014 PMID: 24433356).54

Other less common alterations that have been identified in retinoblastoma tumors include differential

expression of some microRNAs55 (Huang et al 2007 PMID: 18026111) and recurrent single nucleotide

variants/insertion-deletions in the genes BCOR and CREBBP (Kooi et al 2016 PMID: 27126562).56 In

comparison to the genomic landscape of other cancers, retinoblastoma is one of the least mutated.56 (Kooi

et al 2016 PMID: 27126562)

Molecular diagnosis [Hilary]

Strategic testing - Tumor testing first for unilateral/PBL for bilateral

Technologies and techniques

NGS [flow chart of molecular techniques]

Cytogenetic strategies (FISH/microarray)

RNA for discovery and VUS functional studies

Protein studies

The presentation of the patient helps to guide the most optimal strategy for retinoblastoma molecular

genetic testing. If the patient is bilaterally affected, the probability of finding a germline mutation in the

RB1 gene is high (example - 97% detection rate in comprehensive laboratory). For this reason, the most

13

optimal strategy for testing bilateral patients involves testing genomic DNA extracted from peripheral

blood lymphocytes (PBL) first. In rare instances, some patients with isolated bilateral retinoblastoma, the

predisposing RB1 mutation has occurred sometime during embryonal development. In these cases, the

RB1 mutation may only be present in some cells and may not be detected in DNA from PBL. Therefore,

in the event that no mutation is identified in the blood of a bilaterally affected patient, DNA from tumor

should be investigated.57

The situation is different for unilateral patients. Given that approximately 15% of unilateral patients

carry germline mutations, the most optimal strategy for highest detection rate is to first test DNA

extracted from a tumor sample. Upon identification of the tumor mutations, targeted molecular analysis

can be performed on DNA from PBL to determine if the mutation is present is the patient’s germline.

When only the tumor is found to carry the mutations, this information can be very valuable for genetic

counselling, reducing the risk of recurrence in siblings and cousins. In addition, this targeted approach

can allow for a more sensitive assessment of the PBL DNA, which can be useful in the detection of low

level mosaic mutations, more common in unilateral cases (cite).57

Sample preparation impacts the quality of DNA. For best results, fresh or frozen tumor samples

should be taken, as opposed to formalin fixed paraffin embedded tumors, in which DNA is often highly

degraded, making it often too fragmented for use in some molecular diagnostic methods. With regards to

genomic DNA from PBL, it is best to collect whole blood in EDTA, as this anticoagulant has minimal

impact on downstream molecular methods.

Technologies and techniques: Given that there are many ways in which the RB1 gene can be mutated,

several molecular techniques are required to assess for the whole spectrum of oncogenic events.

DNA sequencing: Single nucleotide variants (SNVs) and small insertions/deletions can be identified

using DNA sequencing strategies including Sanger dideoxy-sequencing or massively parallel next-

generation sequencing (NGS) methods (Singh et al 2016 PMID: 27582626; Li et al 2016 PMID: 27155049;

Chen et al 2014 PMID: 24282159).58-60 While both strategies function to produce DNA sequences, NGS

14

has the add advantage of producing millions of DNA sequences in a single run, in contrast to one

sequence per reaction with Sanger. Deciding on which technology to use depends on the clinical question

being asked. When screening family members for a known sequencing detectable RB1 mutation, targeted

Sanger sequencing would be a more cost and time effective strategy as opposed to a screen for an

unknown de novo mutation, where NGS may be the most effective screening strategy. Another added

advantage to NGS is the ability to provide deep sequencing, allowing for a much lower limit of detection

(analytic sensitivity) for identify low level mosaic mutations compared to Sanger sequencing (Chen et al

2014 PMID: 24282159).60 .

Copy number analysis: Large RB1 deletions or duplications that span whole exons or multiple exons

typically cannot be easily detected by DNA sequencing. Instead, techniques including multiplex ligation-

dependent probe amplification (MLPA), quantitative multiplex PCR (QM-PCR) or array comparative

genomic hybridization (aCGH) are often used to interrogate for large deletions (ex. 13q14 deletion

syndrome) and duplications. In addition, these techniques can also be used to identify other genomic

copy number alterations seen in retinoblastoma tumors, such as MYCN amplification. Recently, new

developments in bioinformatics analysis hasnew developments in bioinformatics analysis have created

ways in which NGS data can be interrogated for copy number variants59 (Devarajan et al 2015; Li et al

2016 PMID: 27155049).59,61 While the data is promising,promising; the current limitation of targeted NGS

is that capture efficiency is uneven, which reduces the sensitivity of detecting CNVs in comparison to

conventional methods.

Low levelLow-level mosaic detection: Somatic mosaicism can arise in either the presenting patient or

their parent. Detecting a mosaic mutation can be difficult depending on the individual’s level of

mosaicism. As described in the DNA sequencing section, NGS is one tool that can be used detect low

levellow-level mosaicism (see above). In addition, allele-specific PCR (AS-PCR) is an another strategy

that can be used in situations where the RB1 mutation is known (Rushlow et al 2009 PMID: 19280657).49

15

This strategy involves the generation of a unique set of primers specific to the mutation of interest and

can detect mosaicism levels as low as 1%.

Microsatellite analysis: LOH, MCC, identity,

Methylation analysis: In addition to genetic changes, epigenetic changes have been recognized as

another mechanism of retinoblastoma development. Hypermethylation of the RB1 promoter CpG island

results in transcription inhibition of the RB1 gene and has been identified 10-12% of retinoblastoma

tumors (Richter et al 2003).50 This epigenetic event is thought to only occur somatically and has not been

identified constitutionally in any retinoblastoma patients thus far.

RNA analysis:

Protein studies

Cytogenetic strategies: 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 patient’s suspected of 13q14 deletion syndrome. In parents of 13q14 deletion

patients, karyotype analysis can be used to assess for balanced translocations, which increases the risk of

recurrence in subsequent offspring.

Genetic Counseling (Heather/Hilary)

Importance of high detection rate

Targeted familial testing/prenatal testing, preconception testing

Targeted familial testingtesting1,57 is used: tTo determine if a predisposing RB1 mutation has occurred

de novo, parental DNA from PBL is investigated. Even if neither parent is identified to be a carrier,

recurrence risk in siblings is still increased due to the risk of germline mosaicism. DNA from PBL for all

16

siblings of affected patients should be tested for the proband’s mutation. As well, DNA from PBL for

children of all affected patient’s should also be tested for the predisposing mutation.

If the proband’s mutation was identified to be mosaic (ie postzygotic in origin) in DNA from PBL,

parents and siblings of the proband are not at risk to carry the predisposing mutation. However, the

children of mosaic probandaffecteds should be testedtested, as their risk of inheriting the predisposing

RB1 mutation can be as high as 50% depending on the mutation burden in the probands germline.

When a RB1 mutation has been identified in a family, The Known RB1 mutation of the proband can

be tested in his offspring. Ccouples may consider a number ofmultiple options with respect to planning a

pregnancy. Genetic testing performed early in the course of the pregnancy is available in many countries

around the world. Two early procedures are available: 1) chorionic villus sampling (CVS) and 2)

amniocentesis. CVS is a test typically performed between 11-14 weekswks gestationgestation during

which as sample of the placenta is obtained either by transvaginal or transabdominal approach.

Amniocentesis is a test performed after 16 weeks of gestation whereby as sample of the amniotic fluid is

gathered with a transabdominal approach. CVS has a procedure-associated risk of miscarriage of ~1%.

Amniocentesis has a procedure-associated risk of miscarriage between 0.1-0.5%. Though uncommon,

there is a risk for maternal cell contamination whichcontamination that occurs more frequently with

CVS.62

Results of Ggenetic testing results can be used by the family and health care team to manage the

pregnancy. If a mutation is not identified, the pregnancy can proceed with no further

interventionintervention, as there is no increased risk for retinoblastoma beyond the general population

risk. If the mutation is identified, some couples may consider deciding to stop the pregnancy; other

couples will decide to continue with the pregnancy and appropriate intervention, such as early delivery,

will be put into place to improve outcomes.63

17

Some couples know that they wish to continue their pregnancy regardless of the genetic testing results

and are concerned by the risk of miscarriage associated with early invasive prenatal testing. Where

available, couples can also consider the option of late amniocentesis, performed between 30-34 weekswks

gestation. When amniocentesis is performed late into the pregnancy, the key complication becomes early

delivery rather than miscarriage.62 The risk for procedure-associated significant preterm delivery is low

(<3%). Results of genetic testing will be available with enough time to plan for early delivery when a

mutation has been inherited.

In many countries around the world, the option for prenatal genetic testing is not available. Even

where available, some couples may elect to do no invasive testing during the course of the pregnancy.

For these conceptions, if the pregnancy is at 50% risk for inheriting a RB1 mutation, it is crucial that the

pregnancy does not go post-dates. Induction of labour should be seriously considered if natural delivery

has not occurred by the due date.57,63

Preconception testing

In some countries around the world, there is an in vitro fertilization option available to couples called

preimplantation genetic diagnosis (PGD).64-67 For PGD, a couple undergoes in vitro fertilization.

Conceptions are tested at an early stage of development (typically 8-cell) for the presence of the familial

mutation. Only those conceptions that do not carry the mutation will be used for fertilization. The

procedure is costly, ranging from $10,000-$15,000 per cycle. In some countries, there may be full or

partial coverage of the costs associated with procedure. In addition to cost, couples must consider the

medical and time impact of undergoing in vitro fertilization. Couples also need to be aware that the full

medical implications of PGD are not yet understood; there is emerging evidence that there may be a low

risk for epigenetic changes in the conception as a result of the procedure. For couples that undergo PGD,

it is recommended that typical prenatal testing be pursued during the course of the pregnancy to confirm

the results.64-67

18

Molecular Screening for Retinoblastoma

This can be performed either prenatal or it can be performed at birth via umbilical cord blood

(postnatal screening). This will help either eliminate the 50% theoretical risk of the proband’s RB1

mutation heritability or confirm it into 100% risk. Both screening methods are effective in improving

visual outcome and eye salvage than non-screened children, However, prenatal screening allows for

planning for earlier delivery in positive children (late preterm/early term); this was shown to have less

number of tumors at birth (20% versus 50 %) with only 15 % visual threatening tumors in prenatatl

screening. Prenatal screening with early delivery showed less tumor and treatment burden with higher

treatment success, eye preservation and visual outcome.63

Surveillance for mets and second cancer

Benefits of genetic counsellingcounseling (Table of risk% [skalet etc] [impact new data?] ie: siblings,

offspring, cousins, faroff relatives, stats below population risk]

Genetic counsellingcounseling is both a psychosocial and educational process for patients and their

families with the aim of helping families better adapt to the genetic risk, the genetic condition, and the

process of informed decision makingdecision-making.68-70 (Uhlmann et al. (2009), Shugar (2016)).

Genetic testing is an integral component of genetic counsellingcounseling that results in more informed

and precise genetic counsellingcounseling. Concrete knowledge of the genetic test outcomes results in

specificity, reducing the need for other possible scenarios to be discussed with the family. This enhances

the educational component of genetic counsellingcounseling and also provides further time for

psychosocial support to be provided to the family.

Patients with bilateral retinoblastoma at presentation are presumed to have heritable retinoblastoma

and a RB1 mutation. Genetic testing provides more accurate information about the type of heritable

retinoblastoma and allows for straightforward testing to determine if additional family members are at

risk. Through genetic testing, a patient may be found to have a large deletion extending beyond the RB1

gene as part of the 13q deletion spectrum. Individuals with 13q deletion syndrome are at risk for

19

additional health concerns requiring appropriate medical management and intervention. Results may

reveal a mosaic mutation which indicates that the mutation is definitively de novo; only the individual’s

own children are at risk and no further surveillance or genetic testing is needed for other family members.

The results may find a low-penetrance mutation which indicates the patient is at reduced risk to develop

future tumours. As genetic testing for retinoblastoma becomes more common place and data accumulate,

surveillance of the proband may one day be matched more precisely to the level of risk for new tumours

for individuals with low penetrance mutations.

Patients with unilateral retinoblastoma greatly benefit from genetic testing and counselling.

Approximately 15% of patients with unilateral retinoblastoma will be found to have heritable

retinoblastoma. Correctly identifying these patients can be lifesaving, for both the patients and their

families. Genetic testing companies focused on enhanced detection of RB1 mutations are able to identify

nearly 97% of all retinoblastoma mutations. Genetic testing of the patient’s blood is sensitive enough

when thorough methods are used that not finding a mutation results in a residual risk of heritable

retinoblastoma low enough to remove the need for examinations under anesthesia. This reduces the health

risk for the patient and the cost to the health care system. Testing is even more accurate when a tumour

sample is collected and tested when available. When mutations are identified in the tumour and are

negative in blood, the results can eliminate the need for screening of family members and provide

accurate testing for the patient’s future children. Whether or not a tumour sample is available, finding a

RB1 mutation in a patient’s blood confirms that this patient has heritable retinoblastoma. This patient now

benefits from increased surveillance designed to detect tumours at the earliest stages and awareness of an

increased lifelong risk for second cancers. Members of the patient’s family can have appropriate genetic

testing to accurately determine who is at risk. As with patients with bilateral retinoblastoma, knowing the

specific type of mutation provides the most detailed provision of medical management and counselling.

20

Screening for Retinoblastoma

The Known RB1 mutation of the proband can be tested in his offspring. This can be performed via

amniocentesis during the second trimester of pregnancy with minimal risks on fetus and mother (prenatal

screening) or it can be performed at birth via umbilical cord blood (postnatal screening). This will help

either eliminate the 50% theoretical risk of the proband’s RB1 mutation heritability or confirm it into

100% risk. Both screening methods are effective in improving visual outcome and eye salvage than non-

screened children, However, prenatal screening allows for planning for earlier delivery in positive

children (late preterm/early term); this was shown to have less number of tumors at birth (20% versus 50

%) with only 15 % visual threatening tumors in prenatatl screening. Prenatal screening with early delivery

showed less tumor and treatment burden with higher treatment success, eye preservation and visual

outcome.63

Cost-effectiveness [Brenda/Crystal] {FIGURE/FLOW CHART}

Difficulties and opportunities across different jurisdictions/countries [Jeffry/Sameh]

Compare/contrast Canada vs China vs Jordon

Societal/cultural challenges to GC

In China, many families with retinoblastoma children do not understand the benefits of genetic testing

and genetic counseling in treatment and follow-up. Meanwhile, the health insurance can’t cover the cost

for it. So all the obstacles mentioned above result in the limited application of genetic testing and genetic

counseling nationwide, which also lead to the redundant economic burden on the affected families. The

Chinese government started new policy that allowed every family to have one more child nowadays.

Therefore, genetic testing and genetic counseling should be put into good use especially for the families

carrying the germline RB1 mutation.

21

References

Uhlmann, WR; Schuette, JL; Yashar, B. (2009) A Guide to Genetic Counseling. 2nd Ed. Wiley-

Blackwell.

Shugar, A. (2016) Teaching Genetic Counseling Skills: Incorporating a Genetic Counseling

Adaptation Continuum Model to Address Psychosocial complexity. J Genet Counsel. Epub ahead of

print. PMID: 27891554 DOI: 10.1007/s10897-016-0042-y

Benefits of genetic testing for the proband and family members [Heather]

Prenatal vs Postnatal [Sameh]

Cost-effectiveness [Brenda/Crystal] {FIGURE/FLOW CHART}

Difficulties and opportunities across different jurisdictions/countries [Jeffry/Sameh]

Compare/contrast Canada vs China vs Jordon

Societal/cultural challenges to GC

Conclusions

22

REFERENCES

Uhlmann, WR; Schuette, JL; Yashar, B. (2009) A Guide to Genetic Counseling. 2nd Ed. Wiley-

Blackwell.

Shugar, A. (2016) Teaching Genetic Counseling Skills: Incorporating a Genetic Counseling

Adaptation Continuum Model to Address Psychosocial complexity. J Genet Counsel. Epub ahead of

print. PMID: 27891554 DOI: 10.1007/s10897-016-0042-y

23

Table X:

Subretinal Fluid (RD)

No≤ 5 mm

>5 mm - ≤ 1 quadrant

> 1quadrant

Tum

or

Tumors ≤ 3 mm and further than 1.5 mm from the disc and fovea cT1a/A cT1a/B cT2a/C cT2a/D

Tumors > 3 mm or closer than 1.5 mm to the disc and fovea cT1b/B cT1b/B cT2a/C cT2a/D

Se

edin

g Localized vitreous/ subretinal seeding cT2b/C cT2b/C cT2b/C cT2b/Ddiffuse vitreous/subretinal seeding cT2b/D

High

risk

feat

ures

Phthisis or pre-phthisis bulbi cT3a/ETumor invasion of the pars plana, ciliary body, lens, zonules, iris or anterior chamber cT3b/ERaised intraocular pressure with neovascularization and/or buphthalmos cT3c/EHyphema and/or massive vitreous hemorrhage cT3d/EAseptic orbital cellulitis cT3e/EDiffuse infiltrating retinoblastoma ??/E

Extraocular retinoblastoma cT4/??

clinical T (cT) versus International Intraocular retinoblastoma Classification (IIRC) (cT/IIRC); ?? Not

applicable ; RD Retinal detachment

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

2. 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.

3. Dimaras H, Kimani K, Dimba EA, et al. Retinoblastoma. Lancet. 2012;379(9824):1436-1446.4. Chantada GL, Qaddoumi I, Canturk S, et al. Strategies to manage retinoblastoma in developing

countries. Pediatric blood & cancer. 2011;56(3):341-348.5. Canturk S, Qaddoumi I, Khetan V, et al. Survival of retinoblastoma in less-developed countries

impact of socioeconomic and health-related indicators. Br J Ophthalmol. 2010;94(11):1432-1436.6. 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.

24

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

8. Balmer A, Zografos L, Munier F. Diagnosis and current management of retinoblastoma. Oncogene. 2006;25(38):5341-5349.

9. Munier FL. Classification and management of seeds in retinoblastoma. Ellsworth Lecture Ghent August 24th 2013. Ophthalmic Genet. 2014;35(4):193-207.

10. Balmer A, Munier F. Differential diagnosis of leukocoria and strabismus, first presenting signs of retinoblastoma. Clin Ophthalmol. 2007;1(4):431-439.

11. Gallie BL, Soliman S. Retinoblastoma. In: Lambert B, Lyons C, eds. Taylor and Hoyt's Paediatric Ophthalmology and Strabismus. Vol 5th Edition. Oxford, OX5 1GB, United Kingdom: Elsevier, Ltd.; In Press.

12. 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.

13. Theodossiadis P, Emfietzoglou I, Grigoropoulos V, Moschos M, Theodossiadis GP. Evolution of a retinoma case in 21 years. Ophthalmic Surg Lasers Imaging. 2005;36(2):155-157.

14. Popovic MB, Diezi M, Kuchler H, et al. Trilateral retinoblastoma with suprasellar tumor and associated pineal cyst. J Pediatr Hematol Oncol. 2007;29(1):53-56.

15. Antoneli CB, Ribeiro Kde C, Sakamoto LH, Chojniak MM, Novaes PE, Arias VE. Trilateral retinoblastoma. Pediatr Blood Cancer. 2007;48(3):306-310.

16. 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.

17. 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.

18. Skrypnyk C, Bartsch O. Retinoblastoma, pinealoma, and mild overgrowth in a boy with a deletion of RB1 and neighbor genes on chromosome 13q14. American journal of medical genetics. 2004;124A(4):397-401.

19. 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.

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

21. Knudson AG. Mutation and cancer: statistical study of retinoblastoma. Proceedings of the National Academy of Science, USA. 1971;68(4):820-823.

22. 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.

23. 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.

24. 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.

25. 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.

26. 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.

27. 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.

25

28. Schubert EL, Strong LC, Hansen MF. A splicing mutation in RB1 in low penetrance retinoblastoma. Hum Genet. 1997;100(5-6):557-563.

29. Lefevre SH, Chauveinc L, Stoppa-Lyonnet D, et al. A T to C mutation in the polypyrimidine tract of the exon 9 splicing site of the RB1 gene responsible for low penetrance hereditary retinoblastoma. J Med Genet. 2002;39(5):E21.

30. 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.

31. Cowell JK, Bia B. A novel missense mutation in patients from a retinoblastoma pedigree showing only mild expression of the tumor phenotype. Oncogene. 1998;16(24):3211-3213.

32. 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.

33. Bunin GR, Emanuel BS, Meadows AT, Buckley JD, Woods WG, Hammond GD. Frequency of 13q abnormalities among 203 patients with retinoblastoma. J Natl Cancer Inst. 1989;81(5):370-374.

34. Mitter D, Ullmann R, Muradyan A, et al. Genotype-phenotype correlations in patients with retinoblastoma and interstitial 13q deletions. Eur J Hum Genet. 2011;19(9):947-958.

35. Matsunaga E. Retinoblastoma: host resistance and 13q- chromosomal deletion. Hum Genet. 1980;56(1):53-58.

36. Albrecht P, Ansperger-Rescher B, Schuler A, Zeschnigk M, Gallie B, Lohmann DR. Spectrum of gross deletions and insertions in the RB1 gene in patients with retinoblastoma and association with phenotypic expression. Hum Mutat. 2005;26(5):437-445.

37. Lohmann DR, Brandt B, Hopping W, Passarge E, Horsthemke B. Distinct RB1 gene mutations with low penetrance in hereditary retinoblastoma. Hum Genet. 1994;94(4):349-354.

38. 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.

39. 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.

40. 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.

41. 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.

42. Nevins JR. The Rb/E2F pathway and cancer. Hum Mol Genet. 2001;10(7):699-703.43. Cobrinik D. Pocket proteins and cell cycle control. Oncogene. 2005;24(17):2796-2809.44. Sage J, Cleary ML. Genomics: The path to retinoblastoma. Nature. 2012;481(7381):269-270.45. Knudsen ES, Knudsen KE. Tailoring to RB: tumour suppressor status and therapeutic response.

Nat Rev Cancer. 2008;8(9):714-724.46. Xu XL, Singh HP, Wang L, et al. Rb suppresses human cone-precursor-derived retinoblastoma

tumours. Nature. 2014;514(7522):385-388.47. Lohmann DR. RB1 gene mutations in retinoblastoma. Hum Mutat. 1999;14(4):283-288.48. McEvoy J, Nagahawatte P, Finkelstein D, et al. RB1 gene inactivation by chromothripsis in

human retinoblastoma. Oncotarget. 2014;5(2):438-450.49. Rushlow D, Piovesan B, Zhang K, et al. Detection of mosaic RB1 mutations in families with

retinoblastoma. Hum Mutat. 2009;30(5):842-851.50. 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):253-269.51. 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.

26

52. Munier FL, Thonney F, Girardet A, et al. Evidence of somatic and germinal mosaicism in pseudo-low-penetrant hereditary retinoblastoma, by constitutional and single-sperm mutation analysis. Am J Hum Genet. 1998;63(6):1903-1908.

53. 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.

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

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

56. 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.

57. 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.

58. Singh J, Mishra A, Pandian AJ, et al. Next-generation sequencing-based method shows increased mutation detection sensitivity in an Indian retinoblastoma cohort. Mol Vis. 2016;22:1036-1047.

59. 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.

60. 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.

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

62. 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 in obstetrics & gynecology : the official journal of the International Society of Ultrasound in Obstetrics and Gynecology. 2015;45(1):16-26.

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

64. Dhanjal S, Kakourou G, Mamas T, et al. Preimplantation genetic diagnosis for retinoblastoma predisposition. Br J Ophthalmol. 2007;91(8):1090-1091.

65. Dommering CJ, Moll AC, Imhof SM, de Die-Smulders CE, Coonen E. Another liveborn after preimplantation genetic diagnosis for retinoblastoma. Am J Ophthalmol. 2004;138(6):1088-1089.

66. 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.

67. Girardet A, Hamamah S, Anahory T, et al. First preimplantation genetic diagnosis of hereditary retinoblastoma using informative microsatellite markers. Mol Hum Reprod. 2003;9(2):111-116.

68. 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.

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

70. Shugar AL, Quercia N, Trevors C, Rabideau MM, Ahmed S. Risk for Patient Harm in Canadian Genetic Counseling Practice: It's Time to Consider Regulation. J Genet Couns. 2016.

27