•

CDKN2A/ and familial cancer

Sophie Sun Department of Biology McGili University, Montréal • September 1996

A thesis submitled to the Faculty of Graduate Studies and Research in partial fulfillment of the requirements for the Master's degree in Science

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Canada • AB5TRACT CDKN2A/p16 is a inhibitor which blacks abnormal ccli growth and

proliferation. The CDKN2A is frequently mutated or deleted in a wide variety of

tumour types. Germline mutations have also been identified in familial atypical

multiple mole (FAMMM) pedigrees. However, the role of CDKN2A in

hereditary cancer is uncertain. To explore the relationship between CDKN2A germline

mutations and risk of cancer, 75 families with cancers at multiple sites were analysed

for germline mutations in the CDKN2A gene. A Met53I1e mutation was found in a

non-FAMMM kindred with multiple cancers, including one case of melanoma. The

Met53Ile mutation has been previously reported in three Australian FAMMM kindreds.

A known Ala148Thr polymorphism was also detected in 5 individuals. No other

families were found to have CDKN2A alterations. There were no reported CDKN2A

mutations in families without cases of melanoma. Analysis of microsatellite markers

adjacent to CDKN2A on 9p21 revealed that this family shares a common

haplotype with one other family with this mutation, suggesting that Met5311e is a

founder mutation. These results suggest that while CDKN2A mutations are not

restricted to FAMMM pedigrees, they are very rare or absent in families with

individuals without melanoma. • SOMMAIRE CDKN2A/pI6 est un inhibiteur du cycle cellulaire qui a la fonction d'empêcher

une croissance anormale de la cellule ainsi que sa prolifération. Le gène CDKN2A est

fréquemment muté ou délété dans une large varieté de types de tumeur. Des mutations

germinales furent aussi identifiées dans des familles ayant mélanome familiale (familial

atypical multiple mole melanoma, FAMMM). Cependant, le rôle defini de CDKN2A

dans le cancer héréditaire n'est pas connu. Afin de mieux observer la relation entre les

mutations germinales CDKN2A et le risque de développer le cancer, les mutations

germinales CDKN2A de 75 familles présentant des cancers à sites multiples furent

analysées. Une mutation Met53Ile fut trouvé dans une famille présentant un cas de

mélanome et à cancers multiples. Cette mutation fut déjà connu fut aussi découvert chez

5 individus. Aucune autre famille n'a présenté une altération du gène CDKN2A. De plus,

les familles sans mélanome n'ont d:5montré aucune mutation de CDKN2A. Après une

analyse des marquer microsatellites adjacent au gène CDKN2A sur le chromosome 9p21,

il fut trouvé que cette famille partage un haplotype commun avec une autre famille

australienne ayant la mutation, donc il semble que la mutation Met53Ile ait le même

origine dans deux familles. Ces résuitats suggèrent que les mutations CDKN2A ne sont

pas limitées aux familles FAMMM, mais les mutations sont rares ou absentes dans des

familles sans cas de mélanome.

iii • ACKNOWLEDGEMENTS First and foremost, 1 would like to thank the fanlilies for generously participating in this work.

ln particular, 1thank William Foulkes for his help, guidance and support throughout ail my endeavours. 1would like to acknowledge my advisors David Rosenblatt. Steven Narod, Guy Rouleau, Roy Gravel, Barid Mukherjee and Patricia Tonin for thcir critical assessment ofthis work; my collaborators Serge Jothy. Nick HaywarJ. Ben Milncr. Donald Black and Graeme Walker; Sepideh Karimi for her technical. moral and tinancial assistance; Tamar Flanders for her contribution to the tables and ligures; to my collcagucs and friends at the Division ofMedical Genetics. both past and present: Alex Liede. Chia Chia Sun, Cathy Phelan, Kiran Dole, Marie-Claude Faucher, Hatida Lounis, and France Dion, 1 consider myselfvery fortunate to have worked with a group ofspirited and talented people. This work was supported by the Fonds de Recherche en Santé du Québec.

jv • ABBREVIATIONS Ala alanine bp base pairs CDK cyclin-dependent kinase CDKI cyclin-dependent kinase inhibitor CDKN2A cyclin-dependent kinase inhibitor 2A dATP deoxyadenosine tri-phosphate DNA deoxyribonucleic acid FAMMM familial atypical multiple mole melanoma Ile isoleucine kb kilobase LiCI lithium chloride LOH loss of heterozygosity Mb megabase Met methionine NaOH sodium hydroxide PCR polymerase chain reaction SSCP single-strand conformation polymorphism • Thr threonine

v TABLE OF CONTENTS • Title i Abstract ii Sommaire iii Acknowledgements iv Abbreviations v Table of Contents vi List of tables viii List of figures ix

Chapter 1 INTRODUCTION 1.1 Cancer

1.2 Hereditary cancer 2 a) The 'two-hit' hypothesis 3 b) Genetic predisposition to cancer: identifying individuals at risk 4

1.3 Cell Cycle Control 5

1.4 Cyclins and Cyclin-dependent kinases (CDKs) 5

1.5 Cell Cycle Inhibitors 8 a) CDKN1 9 b) CDKN2 proteins 10

1.6 CDKN2A and cancer 11

1.7 CDKN2A and hereditary cancer 14 a) Familial melanoma 14 b) Germline CDKN2A mutations 15

1.8 Purpose of this study 19

Chapter 2 MATERIALS AND METHOOS

2.1 Cancer families 20 a) Group 1 21 b) Group 2 21 c) Group 3 22 d) Group 4 22

vi 2.2 DNA Extraction 23 a) From Lymphocytes 23 • b) From Paraffin Blocks 24 2.3 Mutation Screening 25 a) PCR Amplification 25 b) Beads Reaction 26 c) Direct Sequencing 26

2.4 Characterisation/Confirmation of Mutations 28 a) Restriction Enzyme Digestion 28 b) PCR-SSCP Analysis 28 c) PCR-Microsatellite Analysis 29

Chapter 3 RESULTS

3.1 Mutation Analysis 30

3.2 Restriction Enzyme Digestion 32

3.3 SSCP Analysis 33

• 3.4 Haplotype Analysis 34

Chapter4

DISCUSSION 36

SUMMARY 47

CLAIMS TO ORIGINALITY 48

PUBLICATIONS 49

REFERENCES 50

APPENDIX A: Pedigrees 61

vii • LIST OF TABLES

2.1 Families for CDKN2A Mutation Analysis

2.2 Primer Sequences for CDKN2A Mutation Analysis

2.3 Primer Sequences for CDKN2A Haplotype Analysis

3.1 Summary of Mutation Analysis Results

4.1 CDKN2A Somatic Mutations in GeU Lines

4.2 CDKN2A Somatic Mutations in Primary Tumours

viii • LIST OF FIGURES

1.1 CDKs, cyclins and the cell cycle

1.2 CDKN2 proteins and the cell cycle

1.3 Germline CDKN2A alterations

3.1 a) Met53l1e-sequencing results b) Met53I1e-SSCP results

3.2 Pedigree offamily 231

3.3a) Ala148Thr-sequencing results b) Ala148Thr-sequencing results

3.4 Results fram 9p21 haplotype analysis

3.5 Results fram haplotype and mutation analyses of familieswith Met53l1e: 231,41001,60001

4.1 Alignment of amine acid sequence of p16cDKN2A pratein family

4.2 Structure of methionine and isoleucine

ix Chapter 1 • INTRODUCTION

1.1 Cancer

It is estimated that one in three people will develop cancer over a lifetime. One in

five are expected to die t'rom maIignant disease. Approximately 100.000 new cases of

l cancer are reported in Canada every year • thus cancer represents a significant burden to

the health care system.

Cancer is a complex family ofdiseases where the fundamental rules ofcell

behaviour break down. Whereas normal cell growth is carefully regulated to meet the

needs ofthe whole organism, cancer cells evade normal controls on proliferation and • spread, and follow their own internai agenda. For cancer to develop. numerous ditTerent control mechanisms have to be bypassed or disrupted by aberrations in key gcncs

important for maintaining cellular integrity. In particular, two gene classes play major

roles in cancer: normally involved in encouraging cell growth (proto-oncogcnes)

and those inhibiting it (turnour suppressor genes).

When mutated, proto-oncogenes can become carcinogenic oncogenes that drive

excessive cell proliferation. For example, the ras family ofgenes encode proteins

transrnitting signaIs t'rom growth factor receptors to other proteins, stimulating cells to

grow. Oveructivation ofmutant ras genes triggers cells to replicate autonomously and

continuously, becoming cancerous. Notably, hyperactive Ras proteins are found in about

25% ofall human tumours, including carcinomas ofthe colon, pancreas and lung. In addition to overstimulation ofgrowth-promoting machinery, the inactivation of • genes providing inhibitory signais to cell growth is commonly observed in many types of malignancies. For example, the tumour suppressor gene TP53 is mutated in over 50% of

ail human cancers. The encoded by this gene is has a critical role in cell cycle

control, sensing abnormalities in several different cellular processes, resulting in cell

cycle arrest (and sometimes cell death). Loss offunction ofthe TP53 gene product can

have catastrophic consequences for the cell. It is therefore not surprising that germline

mutations in this gene lead to the inherited cancer syndrome ofLi-Fraumeni, where early­

onset cancers at many sites occur.

1.2 Hereditary Cancer

The overall risk ofdeveloping cancer is dependent on both environrnental and

genetic factors, although most cancers are probably attributable to exposure to

environrnental carcinogens such as cigarette smoke, chemicals and radiation.

Nonetheless, epidemiological studies ofmany human cancers have demonstrated a

modest two to three-fold increase in risk ofcancer among first-degree relatives with

cancer at the same site. In a sma\l proportion ofcancer cases (probably no more than 5­

10%), genetic factors may he the primary determinant. While the familial clustering of

cancer has been observed for many years, until quite recently, it was net appreciated that

the occurence ofcancer in a proportion offamilies with excessive aggregation ofcancer

could best be explained inheritance ofmutant tumour suppressor genes predisposing to

cancer.

2 al The 'two-hit' hypothesis • In the 1950s and 60s, epidemiological studies ofthe age ofonset ofhuman cancers first suggested that human tumourigenesis was a multistep process, with between

2 two and seven successive mutations needed for a tumour development -4. From a

statistical analysis ofthe paediatric eye tumour, retinoblastoma, it was postulated that

hereditary cancers were caused by the same mechanism as sporadic cancers. In the case

ofhereditary retinoblastoma, the first mutation occured in the germline, and was followed

by mutation ofthe remaining allele in the somatic tissue (ie. the tissue where the cancer

arosei,6. In the case ofsporadic retinoblastoma, il was suggested that both ofthe 'two

hits' occured in the retinoblasts themselves. This hypothesis could exp!ain the earlier age

ofonset associated with bilatera! tumours as compared to unilateral retinob!astomas as

weil as the observation that bilateral tumours were more common in those with a family • history than in those without such a history: the first mutation was already present at conception.

The cloning ofthe retinob!astoma gene in 19867 confirmed Knudson's hypothesis,

although the 'two-hit' theory does not appear to app!y to ail other cancer susceptibility

genes. Nevertheless, this mode! has formed the rationale behind many ofexperiments

100king for candidate tumour suppressor genes, including CDKN2A, b) Genetic predisposition to cancer: identifying at-risk individuals

In general, heritabIe and non-heritable tumours are indistinguishable from each

other, and the diagnosis ofhereditary cancer is made on the basis ofpersonaI and family

histories. A genetic cause ofcancer is suggested by an early age at onset, multiple

tumours or bilateral disease, and a pattern offamilial clustering compatible with

autosomal dominant inheritance. For example, breast cancer appears at a much earlier age than expected in both the hereditary breast-ovarian cancer and Li-Fraumeni

syndromes. Sorne tumour types are associated with pre-neoplastic lesions in adjacent

tissues or with other characteristic benign features. Multiple colonic polyps with colon cancer is typical offamilial adenomatous polyposis (FAP). The presence ofgeneralized

C-cell hyperplasia with a medullary thyroid carcinoma is suggestive ofmultiple endocrine

neoplasia type 2A (MEN2A). For a few well-defined inherited cancer syndromes,

individuals may be recognized by visible physical characteristics. For example, the

presence ofmultiple dysplastic naevi are diagnostic features offamiliai atypical multiple

mole melanoma syndrome (FAMMM), also known as hereditary dysplastic naevi

syndrome (HDNS).

Multiple primary cancer diagnosed in the same individual may be due to chance,

may be due to common environmental exposure, may be the late effect oftreatment ofthe

first malignancy, or may be due to genetic predispostion. A high proportion ofchildren

with both retinoblastoma and osteosarcoma carry mutations ofRBl. Similarly, survivors

ofchildhood adrenocortical cancer are at a high risk ofdeveloping a second childhood

cancers. As a consequence ofthe identification ofindividuals with inherited cancer,

4 presenting key features such as bilateral tumours, young age ofonset, and multiple • primaries, an increasing number ofcancer susceptibility genes are being mapped ,md cloned. In particular, a category ofthese genes have been found to encode components

involved in cell cycle control.

1.3 Cell Cycle Control

Recent advances in the understanding ofthe eell cycle have provided insights into

molecular mechanisms for malignant transformation by deregulation ofthe ccll cycle.

The cell cycle is a fundamental biological process that enables the cell to grow, dividc and

separate. Il can be divided into four phases: 01, during which the cells begin to grow:

followed successively by S, the period when DNA synthesis occurs as the ccll repl icatcs

its ; a second growth phase, 02; and finally cell division or mitosis, M.

• Transitions through the different phases or 'checkpoints' are tightly controlled by series

ofprecisely timed events involving families ofproteins that include the cyclins, cyclin­

dependent kinases (CDKs) and the recently identified cyclin-dependent kinase inhibitors

(CDKIs).

1.4 Cyclins and Cyclin-dependent kinases (CDKs)

Much ofour present knowledge ofcell cycle regulation in mammals hascome

from early studies in yeast, sea urchins, and frogs. In the 1970s, Hartwell and his

colleagues identified mutants in the budding yeast, Saccharomyces cerevisiae. that were

9 defective at different stages ofthe cell cycle . These mutants were found to carry mutations in a series ofgenes called CDC genes, encoding proteins important in allowing cell cycle progression. One particular gene product, CDC28, appeared to control entry into mitosis. Nurse and Bisset (1981) later identified a gene, CDC2, in the fission yeast,

Saccharomyces pombe, which was shown to be functionally homologous to the CDC28

lO gene in S. cerevisiae • Subsequently, a cytoplasmic factor, called maturation promoting factor (MPF), was isolated in unfertilised frog (Xenopus) eggs by its ability to induce immature oocytes to undergo mitotic division. MPF was found to be a protein complex containing a protein identical to the CDC2 gene product ofS. pombeIl .

Furthermore, Hunt and his colleagues were studying patterns ofprotein synthesis during development in fertilised sea urchin eggs when they found one protein ofabout

12 55kDa that was transiently expressed during the division cycle • Notably, the IeveIs of this protein, which they termed 'cyclin', built up during interphase ofmitosis and was absent when the egg divided.

These studies provided clues to potentiaI key players involved in cell division, and crucial evidence linking MPF, COC2 and cyclin together came from experiments that followed. In addition to containing COC2 protein, purification ofMPF from frog eggs revealed the presence ofa cyclin called cyclin B. It was shown that the variable expression ofcyclin B was tied to G2 to M progression. COC2 was also found to be have protein kinase activity that osciIlated with cell cycle phase in a cyclin-dependent manner.

Similarly, transiently expressed cyclins in the yeast S. cerevisiae were identified (called

CLN 1,2 and 3) and were shown to bind to and activate the COC2 homologue, COC28 kinase, driving the cell from one stage to another. A model ofeukaryotic cell cycle

6 control gradually emerged: progression through transitions or 'checkpoints' bctween • different cell cycle states is driven by a family ofprotein kinases, the cyclin-dependent kinases (CDKs) and their obligate activating partners, the cyclins.

Functional homologues ofthe yeast cyclins and CDKs have since been isolated in

mammals and their similarity is striking; most ofthe mammalian cyclins and CDKs can

functionally replace the corresponding yeast proteins. Mammalian cells contain at least

11 cyclins (A, BI, B2, C, Dl, D2, D3, E, F, G and H) and 5 CDKs (CDC2, CDK2.

CDK4, CDK6 and CDK7) whereby specific cyclin/CDK complexes regulate different ccli

cycle checkpoints (Figure 1.1).

One ofthe most important transitions occurs in late GI,just before the star! of

DNA replication. This checkpoint is known as START in yeast or as the restriction point

in mammalian cells at which the cell commits itselfto another round ofDNA replication

and at which both positive and negative external signais are integrated into the ccli. The

D-type cyclins associate with and activate the CDK4/6 kinases, allowing the cell to

progress through the restriction point. After the cell has passed the restriction point the

cyclin E/CDK2 complex forrns, initiating DNA replication. Cyclin A/CDK2 is required

continuously for progression through , and also for G2/M transition. Finally,

entry into mitosis is signaled by activation ofcyclin B/CDC2 complex.

Deregulation ofcell cycle checkpoints has been linked to cancer, often through

changes in the cyclin/CDK complexes. ln sorne instances, this is due to disruption in the

cyclin genes themselves. Alterations that cause the overexpression ofthe D-type cyclins

are commonly observed in different cancers. The CCNDI (or PRADI) gene on

7 •

FIGURE 1.1 Diagram showing the involvement of human cyclin- . dependent kinases (CDKs) and cyclins during the different phases (G1, 5, G2, M) of the cell cycle. Abbreviations include: : transcription factor PCNA: proliferating cell nuclear antigen R point: restriction point •

CDKs, cyclins and the cell cycle chromosome IIq13 encoding cyclin 01 has been identified as a proto-oncogene that can • be overexpressed via numerous aberrations such as gene amplification, chromosomal l3 rearrangements, provirai insertion, and other mechanisms • AnaIogous abnormaIities can

also activate cyclin 02, and cyclin E levels are aItered in sorne tumours14.

Aberrations in genes encoding the COKs, the cyclin partners, have aIso been

reported in human cancer. For example, CDK4 amplification has been described in

l5 approximately IS% ofanaplastie astrocytomas and • Wolfel et al. 16

(199S) found a R24C somatic CDK4 mutation in 2/29 . The same mutation

was reported in the germline oftwo apparently unrelated melanoma families and shown

17 to segregate with the disease • The R24C mutation produces a COK4 protein unable to

bind pl6COKN2A and plSCOKN2B (COK4 inhibitor proteins, discussed below) 16.

ln addition, the CDK6 gene localises to chromosome band 7p21 18, a region that is

l9 commonly rearranged in melanoma celllines and tumours • Hence, these results suggest

that the disruption ofthe cyclins and COKs, which act as positive regulators ofthe cell

cycle, are important events during tumourigenesis.

1.5 Cell Cycle Inhibitors

Recently, a new type ofcell-cycle regulator, the cyclin-dependent kinase

inhibitors (COKIs) have been identified and they include p21, p27, pS7, piS, p16, p18,

and p19. These proteins act as negative regulators ofthe cell cycle by binding and

inhibiting the activity ofthe cyclin/COK complexes. The cell cycle inhibitors block

abnormal cell growth and proliferation in response to both extemal and internai signais, thus potentially act as tumour suppressors; loss ofthe restraints imposed on the cell cycle • by the CDK inhibitors can result in continuous cell growth under conditions which would normally induce growth arrest, leading to tumour development.

a) CDKN1 proteins

The CDK inhibitors can be divided into !Wo classes. The tirst group is the

CDKNI family which includes CDKNIA (p2Iio'22 , CDKNIB (P27)23.24 and CDKNIC

(P57)2s.26. Biochemical assays suggest that the CDKNI proteins are global cell cycle

inhibitors, capable ofbinding and inhibiting a wide variety ofcyclin/CDK complexes.

Although these inhibitors behave similarly in vitro, their expression in vivo is under the

control ofdifferent mitogenic and antimitogenic signais. For example, CDKN1A (also

calledp21, CIPI. WAFl, SDIJ, CAnO or PIC]) is transcriptionally induced by the

protein in G1, and it is an important mediator ofcell cycle arrest imposed by p53 in

response to DNA damage. In comparison, CDKNIB (P27KIP1 ) is not a p53 response

gene but is implicated in arrest in response to other extracellular growth

inhibitory signais, such as TGF-~ and cell-cell contact. Although CDKNIA and

CDKN1B are potential targets for inactivation during tumourigenesis, somatic mutations

27 30 in these genes have been rare1y seen in human cancers . • No germline mutations have

been reported so far.

Interestingly, !Wo polymorphisms in the CDKNlA (P21) gene, one in codon 31

27 29 and another in the 3'UTR, have been reported - .31. Il was observed that the presence of

these polymorphisms was correlated with the absence of TP53 somatie mutations in

9 breast cancer and sarcoma patients, implying that polymorphisms might influence • CDKNJA in such a way to obviate the requirement for TP53 mutations to deregulate cell cycle function. However, we have analysed a large series ofovarian tumours and found

no significant difference in distribution CDKNJA polymorphisms in turnours with or

without TP53 mutations. Therefore, the presence ofa variant CDKNJA allele does not

32 appear to have an aetiological role in ovarian cancer •

b) CDKN2 proteins

A second family ofrecentIy isolated cell cycle inhibitors, the CDKN2 proteins,

3 35 include CDKN2A (P16), CDKN2B (PIS), CDKN2C (PIS) and CDKN2D (pI9i - .

These low molecular weight proteins have a high degree ofsequence homology,

containing 4 -Iike repeats, a structural motif shared by other proteins that regulate

36 • the cell cycle and differentiation . The ankyrin domains presumably have sorne role in cell growth and differentiation, and they may be involved generally in protein-protein

37 interactions .

The CDKN2 proteins specifically inactivate the cyclin/CDK4 and cyclin D/CDK6

38 complexes by competing with the D-type cyclins for binding directly to the CDKs •

CDK4 and CDK6 regulate passage through the restriction checkpoint from 01 to S phase.

Cyclin D/CDK4 and cyclin D/CDK6 complexes phosphorylate the retinoblastoma

suscepti.bility gene product, pRb. During 01, Rb protein binds and sequesters specifie

proteins necessary for cell cycle progression, including members ofthe E2F famiIy (E2F

is a transcription factor originally identified by its role as an activator ofthe adenovirus

10 E2 promoter), which are necessary for the expression ofmany genes encoding proteins • involved in DNA replication (DNA polymerase a, prolitèrating cell nuclcar antigcn (PCNA), etc.). Once phosphorylated, pRb releases E2F. thereby allowing transcription

ofE2F-responsive genes and entry ofthe cells into S phase (Figure 1.2). Thus, the

CDKN2 proteins play important roles in G1 checkpoint regulation whereby loss of

functional CDKN2 protein might then result in unregulated CDK4 activity leading to

persistent Rb phosphorylation and consequently, uncontrolled cellular proliferation. In

particular, CDKN2A (P16), the founding member ofthe CDKN2 tamily, is implicatcd in

the development ofmany cancers.

1.6 CDKN2A and cancer

In 1993, Serrano et al. isolated a 16 kDa protein, p16, that was able to bind and

33 inactivate CDK4 in vitro using a yeast two-hybrid protein interaction scrcen .

Subsequently, the gene encoding pl6 was positionally cloned at 9p21 by two groups

looking for candidate tumour suppressor genes in this chromosomal region which is

J8 J9 frequently deleted in many types oftumour celllines • . It has been given diffèrent

names including CDK41, MTSI ,p16, INK4A, and was recently designated as CDKl\·:;."

for m'clin gependent kinase inhibitor 2A by the Organisation

nomenclature committee.

The CDKN2A gene consists ofa 468 coding sequence divided into three

encoding a 16kDa protein (hereafter referred to as pI6CDKN2A) that is 156 amino

acids long. In addition, a fourth 1~ has recently been identified, corresponding to an

Il •

FIGURE 1.2 Schematic diagram showing the involvement of CDKN2 proteins (p15, p16, p18, p19) in cell cycle regulation. Abbreviations include: CDK: cyclin-dependent kinase PCNA: proliferating cell nuclear antigen Rb: retinoblastoma gene product E2F: transcription factor • P: phosphate Tx genes: gene transcription CDKN2: cyclin-dependent kinase inhibitor 2 proteins •

CDKN2 inhibitors and the cell cycle

G1 <ê?~ Restriction /' Point

M s

G2 alternative ptranscript that contains exons 2 and 3 as above, but with a different exon 140. • Although exon 1ppossesses an open reading frame, it does not appear to be translated in 40 vivo • At present, it is unclear whether the ptranscript is important for the biological

function ofthe p16CDKN2A protein.

CDKN2A is frequently homozygously deleted in celllines derived from a variety

oftumours, including melanoma, carcinomas ofthe lung, brain, kidney, breast, and ovary,

38 39 as weil as osteosarcomas and leukemias • • In melanoma celllines where one copy of

the gene is absent, the remaining copy is often mutated, supporting the notion that

CDKN2A is a tumour suppressor, particularly involved in the development of

38 melanoma •

Initially, it was observed that the rate ofCDKN2A point mutations was higher in

celllines as compared to primary tumours with allelic loss at 9p21, raising sorne doubts

concerning the significance ofCDKN2A homozygous deletions and mutations seen in cell

lines. This discrepancy suggested that perhaps another tumour suppressor gene is the

target ofLOH in the region, or simply CDKN2A is important for the maintenance of

viability in cell culture. However,!Wo recent findings support the raie ofCDKN2A as a

tumour suppressor involved in many cancers: firstly, Cairns et al. (1995) demonstrated

that CDKN2A microdeletions (spanning less than 200 kilobases, including CDKN2A) are

present in many tumour types. They are only detectable using an array ofmicrosatellites

close to the gene, and have been confirmed by fluorescent in situ hybridisation (FISH)41.

Secondly, CDKN2A is frequently inactivated by 5' CpG methylation in lung cancers,

12 gliomas, head and neck squamous cell carcinomas, and breast cancers, prostate cancers, • renal cancers, bladder cancers, and colon cancers42-44. Although a wide variety of neoplasms exhibit CDKN2A inactivation by

hemizygous and homozygous deletions, as well as by hypermethylation ofthe gene,

intragenic mutations are seen in a significant, yet relatively smaller proportion, oftumours

and celilines. The subset ofcancers with frequent somatic point mutations (nonsense,

missense, frame-shift and splice mutations) include melanomas, non-small celliung

cancers, oesophageal carcinomas, head and neck cancers, pancreatic adenocarcinomas,

45 6 and biliary tract çancers .4 • It is interesting to note that CDKN2A somatic point

mutations are rare in other malignancies. For example, homozygous deletions are found

in 60-65% breast tumours and celilines38.41, but only 2 mutations in primary tumours

7 have been reported thus far45.4 •

While CDKN2A is most commonly inactivated by homozygous deletions in

nurnerous tumour types, the lack ofintragenic CDKN2A mutations in certain cancers

suggests that another gene nearby might be a target for 9p21 deletions contributing to the

progression ofthese malignancies. One good candidate is the CDKN2B (PIS) gene which

is located within 20kb ofCDKN2A. The pl5CDKN2B protein is another CDK4/6

inhibitor that is highly homologous to pI6CDKN2A, sharing a region of81 amino acids

with 97% identity34.

Deletions ofCDKN2A alone, CDKN2B alone, and codeletion ofthe two genes

have ail been described. CDKN2A is usually, but not always, the target of9p21

13 48 deletion • Jen et al. suggest that both genes together are the target ofthe deletions since the two proteins have similar biochemical functions, and the region ofhomozygous deletion most often contains both genes48. An alternative explanation to the 'double target' hypothesis is that the target gene can be CDKN2A in sorne cases, and CDKN2B in others. It is also possible that other as yet unidentified tumour suppressor genes are present in the region. A clearer interpretation ofthese observations awaits further mutation analysis and functional studies ofthese genes, especially CDKN2B.

1.7 CDKN2A and hereditary cancer

A for one type ofgenetic predisposition to melanoma, MLM2, has been mapped to 9p21, implicating CDKN2A as a melanoma susceptibility gene in this region.

a) Familial melanoma

The first documented case ofmelanoma occuring in a familial context is credited to Norris (1820) who described a father and son with atypical multiple moles, both affected with cutaneous malignant melanoma. There were no further reports until the contemporary study by Cawley et al. (1952) who described malignant melanomas in a father and two ofhis three children 49. Subsequently, the identification ofother melanoma kindreds highlighted the role ofheredity in the aetiology ofthis cancerso. In

1978, Lynch et al. sldefined an hereditary disorder, FAMMM-familialntypical multiple­ mole melanoma (also termed hereditary dysplastic naevi syndromeS2 (HDNS) and atypical mole syndromeS3 (AMS», which is characterised by an autosomal dominantly

14 inherited predisposition to cutaneous malignant melanomas in association with multiple • atypical naevi (Iesions thought to represent a precursor to malignant melanoma). It has since been estimated that approximately 8-12% ofcutaneous malignant melanomas are

54 attributable to FAMMM •

ln the early 1990s, molecular cytogenetic studies revealed frequent heterozygous

and hemizygous deletions of9p2l-22 in melanoma celllines and tumours, providing the

tirst evidence that a melanoma susceptibility gene, designated MLM2, resided in this

55 region ,56. Subsequently, linkage studies ofFAMMM kindreds from different

populations detined 9p2l (between the loci ofIFNA-l and D9S126) as the probable

57 60 location ofa tumour suppressor involved in melanoma predisposition • . CDKN2A was

also found to map to tbis region of9p2l and the gene was observed to be frequently

altered in many types ofcelllines and tumours, including melanoma. These findings

suggested that CDKN2A might be MLM2. To test the candidacy ofCDKN2A as the 9p21

familial melanoma gene, several melanoma-prone families were analysed for

predisposing CDKN2A mutations.

b) Germline CDKN2A mutations

ln 1994, shortly after the c10ning ofthe CDKN2A gene, Hussussian el al.

described six CDKN2A germline mutations seen in 9 of 18 American FAMMM

61 kindreds • These alterations included 4 missense mutations, one nonsense and one splice

mutation, wbich were identified in 33/36 melanoma cases. Another two missense

changes (Ala148Thr, Ile4l Thr) did not segregate with melanoma in these families and

15 were detected in nonnal controls, thus considered to be polymorphisms. Ali but one of these families with CDKN2A mutations had a >0.70 probability ofbeing linked to 9p.

A simultaneous study by Kamb et al. reported CDKN2A mutations (Glyl0ITrp,

Val126Asp, also seen by Hussussian et al.) in only 2 out of8 melanoma kindreds although all but one ofthese families were previously shown to be linked to 9p by

62 haplotype analysis • However, five Dutch kindreds in this cohort that were initially described as having no mutations were later found to share a 19bp deletion within exon 2

63 ofCDKN2A •

A regulatory mutation has also been identified in a family first reported by

Hussussian et al. and presumably results in abrogated expression from one CDKN2A

64 allele • Furthennore, Goldstein et al. 64 reported a 24bp duplication mutation in a V.S. family that leads to an iteration ofthe first 8 amino acids ofpI6CDKN2A. This 24bp 65 repeat has also been seen in an Australian melanoma kindred and a family from the V.K. as well (N.Spurr, persona! communication). In the same study ofmelanoma families in

Australia, Walker et al. reported an additiona! six CDKN2A mutations in 7118 kindreds analysed. These included 4 single basepair changes and 1 nonsense mutation.

Another group analysed a set ofAustralian melanoma kindreds where approximately one-third showed possible linkage to 9p and revealed a CDKN2A missense mutation (Arg24Pro) in 1117 families studied. This mutation was found in the only

family that could confidently be assigned to 9p (lod >1.0)66.

16 Liu et al. have described a Canadian melanoma family with a 6bp in-frame deletion resulting in the elimination ofamino acids Leul04 and AsnIOS. Furthermore,

73 the mutant protein was shown to be unable to bind CDK4 in vitro •

An in-frame 3bp duplication resulting in the insertion ofan arginine residue at

codon 113 was identified in two melanoma kindreds from Southem Sweden. The

observation that both families share a common haplotype suggest a founder effect74. In

another study in Sweden, Hansson et al. analysed 101 hereditary melanoma families for

CDKN2A mutations and 4 apparently unrelated kindreds were found to harbour the same

3bp insertion (ins Il1Arg) in exon 2 ofCDKN2A. A Pr048Leu missense change in exon

1 was detected in anothcr Swedish melanoma family67.

Whelan et al. have identified a previously reported Gly 101Trp mutation in an

Arnerican family with melanomas, as weIl as and squamous cell

72 carcinoma ofthe tongue in mutation carriers • Similarly, Ciotti et al. detected the

Giy101Trp mutation in 7 Italian FAMMM kindreds with an increased risk ofpancreatic

cancer71 . In addition, a 12bp in-frame deletion eliminating codons 96-99, shown to

produce a functionally abnormal p16CDKN2A protein, has been reported in a V.S.

kindred affected with melanoma, as weIl as non-small ceIllung cancers and head and

neck cancers.

Biochemical studies oD missense mutations (Glyl01Trp, ValI26Asp, Arg87Pro)

have confirmed that particular disease-related CDKN2A alleles encode functionally

abnormal pl6CDKN2A proteins with impaired ability to bind and inhibit the catalytic

77 activity ofthe cyclin DlICDK4 and cyclin Dl/CDK6 complexes in vitro • Published

17 reports ofin-frame deletions and insertions (deI 96-99, deI 104-105, inslllArg, • insl13Arg) have also demonstrated that these mutations encode mutant pl6CDKN2A 67 proteins with abrogated function ,70,73,74, Other CDKN2A mutations that cause premature

termination through nonsense substitutions (Arg58STOP) or frameshift mutations (I9bp

deI225-243) are expected to yield impaired or defective pl6CDKN2A proteins, In

addition, a common AI48T polymorphism, seen in both melanoma-prone families and

77 controls, was found to behave similarly to wild-type CDKN2A ,78, These results have

been consistent with existing data from families with these mutations, with a few

exceptions,

One melanoma-associated change (Asn71 Ser) did not have reduced affinity for

CDK4 and only marginally differed from wild-type pl6CDKN2A in its ability to inhibit

77 CDK4-mediated phosphorylation ofthe Rb protein , Subsequently, Reymond and Brent

reported similar results, implying that this isoform may be a naturally occuring neutral

78 polymorphism , Conversely, a pl6 variant, Ile49Thr, seen in cancer kindreds as weil as

in the control population and thought to be a non-deleterious polymorphism, was

78 observed to be deficient in interaction with CDK4 and CDK6 ,

ln SUffi, twenty-six germline alterations have been identified in the CDKN2A g<:n<:

so far (see Figure 1.3), These include one regulatory mutation, a splice mutation, 2

nonsense mutations, 7 deletions/insertions, and Il missense mutations that segregate with

melanoma in 59 families from the United States, Australia, the Netherlands, the U,K..

Italy, Sweden and Canada6I,63,65.67,70,73,75.76. Four naturally occuring CDKN2A

polymorphisms have been also described. Recent functional analyses ofmelanoma-

18 •

FIGURE 1.3 Germline CDKN2A alterations identified to date (July 1996) and their relative frequencies. Each symbol to the left of the diagram represents one family in which the corresponding mutation has been found. Underlined mutations occur in the ankyrin consensus sequence. Mutations in bold are also • seen somatically in primary tumours and/or cell lines. CDKN2A Germline Alterations • BASE EVENT POPULATION REF regulatory sequence regulatory USA 64 mutation 24bp ins -1e.8 extra 8 aa's Australia. USA. 64,65, N.Spurr, personal •••• UK communication, G. Walker, personal communication

46 dei C frameshift Australia 6S

71 G>C Arg24Pro Australia 66

EXON 1 9ST>C Leu32Pro Australla 6S ·• 104G>C Gly35A1a Australia 6S 143C>T Pr048Leu SWeden 67 0 146T>C 1Ie49Thr USA 61 · 149A>G Gln50Ars Australia 65

159G>C Met5311e Australia, 65,68, G. WaU:er, personal .... Canada communication

172C>T ArgS6STOP USA 61

212A>G Asn71Ser USA 61 ••••••••••••• , 9bp dei 22S·243 frameshift Netherlands 63 • 14bp dei 240-253 frameshirt USA 69 EXON2 260G>C Arg87Pro USA 61 • 12bp dei 268-279 dei 96-S9 USA 70 • ...... 301 G>T Gly101Trp USA, Ilaly 61,62,71,72 • 6bp dei 31D-31S de1104-10S Canada 73

322 G>A Asp10SAsn Australia G. Walker, personal communication •••• 3 bp ins 331-333 ins 111Arg SWeden 67 3 bp ins 337-339 ins 113Arg 74 •• * - SWeden ... 377T>A Val126Asp USA 61,62 442G>A A1a148Thr USA, Auslralla, 61,62,65,66,74 0 Sweden, Canada · IV52 +1 G>T spllce USA 61 EXON3 soo C>G 3'UTR USA. Australia. 6S,74-76 Sweden :t S40 C>T 3'UTR Australia 66 KEY; • REGULATORY MUTATION • INSERnONIDElETION • MISSENSElNONSENSe o POLYMORPHISM

• Some kindrods with these mutations share a common founder. related mutations have provided biochemical data supporting the involvement of • CDKN2A in cancer predisposition.

1.8 Purpose of this study

Recently, the CDKN2A gene was positionally cloned by groups looking for

candidate turnour suppressor genes on chromosome 9p21, a region that is frequently

deleted in many turnour types. CDKN2A encodes a previously identified cyclin­

dependent kinase inhibitor, p16CDKN2A, that is involved in negatively regulating the cell

cycle, blocking abnormal growth and cell proliferation. The CDKN2A gene is commonly

mutated or deleted in many types ofsporadic cancer and is currently under intense

investigation as a melanoma susceptibility locus. Several germline mutations in the

CDKN2A gene have been identified in familial melanoma (FAMMM) kindreds showing • linkage to 9p and sorne families with other cancers, particularly gastrointestinal cancers and squamous cell carcinoma ofthe head and neck; nevertheless, the spectrum of

turnours associated with inherited CDKN2A mutatiom; remains undefined. This study

aimed to invcstigate the role ofCDKN2A and familial cancer by analysing 75 families

with numerous cases ofcancer at many sites for germline mutations in the CDKN2A gene.

19 Chapter 2 • MATERIAL5 AND METHOD5

2.1 Cancer families

From a resource ofover 300 families that were referred to the Adult Hereditary

Cancer Clinic at the Montreal General Hospital over a three year period, seventy-five

families were selected for CDKN2A mutation analysis based on the criteria that 1)

mutations in other cancer predisposing genes such as BRCAl and TP53 had not been

previously detected in the family and 2) there was a striking family history ofcancers of

many types including melanoma, head and neck cancers, lung cancers, as weil as o carcinomas ofthe kidney, colon, ovary, breast, thyroid, pancreas, prostate, and bladder. The CDKN2A gene is a relatively small gene consisting of468bp divided into 3

exons: 150, 307 and IIbp in size, respectively. The mutation screening strategy involved

PCR amplification and direct sequencing ofCDKN2A exons 1 and 2 (corresponding to

98% ofthe sequence encoding p16CDKN2A) from constitutional DNA from one

individual per family. The families were classified into four groups by the cancer history

ofthe individuals tested, designated the index case, as follows (see Table 2.1; pedigrees in

Appendix A):

20 a) Group 1: Multiple primaries in the index case (n=24) • Twenty-four people with multiple primaries were selected; 11/24 ofthese cases had at least one primary that was melanoma. Two individuals belonging to this subgroup

had multiple primaries ofmelanoma alone with other cancers in the family (210 and 231).

The multi-site cancers in the other thirteen individuals (ranging from 2 to 4 primaries)

included carcinomas ofthe head and neck, breast, colon, the biliary tract, ovary, cervix,

prostate, kidney, and thyroid, as weil as Iymphoma.

b) Group 2: Melanoma in the index and multiple cancers in the family

(n=23)

Most ofthe individuals included in this group did llil1 have a strong tàmily history

ofmelanoma, with the exception offamily 552, where the person tested had two tirst • degree relatives with melanoma. Another four people (from families 191, 192, 196,203) had one other first degree relative with melanoma. The two individuals from families 210

and 231 (who had double me1anoma primaries) were classified in Group 1 as weil. The

remaining 16 families had melanoma in the index with several other cancers elsewhere

such as cancers ofthe breast, colon, lung, biliary tract, cervix, ovary, head and neck,

kidney, brain, prostate, uterus, as weil as lymphoma and 1eukemia.

21 c) Group 3: Other cancer in the index and numerous cancers in the • family (n=28) This group included 2 families with pancreatic cancer, 1 bladder cancer family and

the remaining kindreds had numerous cancers with the index cases diagnosed with

cancers ofthe breast, ovary, colon, prostate, kidney, and thyroid, as weU as non­

Hodgkin's lymphoma and leukemia

d) Group 4: No cancer in the index with many cancers in the family

(n=2)

Two individuals were tested from 2 interesting families, although unaffected, as

blood DNA from affected individuals was unavailable. Family 199 was a kindred

characterised by multiple dyplastic naevi in three individuals, including the person tested.

There were also cases ofbreast cancer, melanoma, and brain cancer reported in other

members ofthe family. Given that family 199 ressembled a FAMMM kindred, it was

included in the CDKN2A mutation analysis.

The index case analysed from family 466 had three sibs who aU died ofcancer.

One sister was diagnosed with breast cancer at age 49, another with pancreatic cancer at

50, and a third sister had lung cancer at 50. The individual tested for mutations had a

benign ovarian cyst. As this person had an extensive family history ofcancer (15/33

affected with cancer in three generations) and only her DNA was available, she was

included in the study.

22 •

TABLE 2.1 Classification of cancer-prone families analysed for CDKN2A germline mutations. Table 2.1 Families for CDKN2A Mutation Analysis

Group No. Classification Family No.

Multiple primaries in index case (n=24) 112 161 210· 214 221 231· 233 239 242 299 306 320 323 324 329 333 365 385 386 438 445 449 479 647

2 Melanoma in index and multiple 157 cancers in family (n=23) 191 192 194 196 197 203 204 207 210 223 228 231 240 249 250 284 295 552 607 N2605 N2621 N2796 Table 2.1 (cont'd) " Group No. Classification FamilyNo.

3 Other cancer in index case and 108 nUmerous cancers in the farr.lly (n=28) 114 117 122 124 126 134 135 138 144 148 152 154 173 195 208 224 259 265 281 296 301 303 305 402 481 559 756

4 No cancer in the index with many 199 cancers in the family (n=2) 466

"also in Group 2; pedigrees in Appendix A 2.2 DNA extraction a) From lymphocytes

Prior to this project, DNA had been extracted from whole blood by using the

protocol described as follows: firstly, to separate plasma, white and red blood cells, the

blood sample was first spun at 1,900 rpm for 10 minutes. The middle layer containing

lymphocytes was transfered to a ISml tube, RSB was added to a final volume of Ilml,

and mixed weil by hand. Ninety microlitres ofNONIDET P-40 detergent (ICN

Biochemicals, the Netherlands) was then added to the tube and mixed weil by inverting.

The sample was spun at 2,SOO rpm for 10 minutes and the supernatant was decanted. The

isolated pellet was resuspended in O.Sml RSB. The cell nuclei were Iysed in 3mls of

sodium dodecyl sulfate (SDS) solution, mixing the suspension gently by repeatedly

inverting the tube. Sixty III ofproteinase K (1 mg/ml) was added and the samples were

incubated overnight at 37oC. Subsequently, DNA was extracted by adding 3ml ofbuffer­

saturated phenol (Gibco-BRL, USA), blending the phases, spinning at 2,SOO rpm for 10

minutes, then discarding the bottom phenol layer. The previous steps were repC)ated

twice, using 3ml phenol-chloroform isoamyl (1:1), followed by 3ml chloroform isoamyl

only. Once the bottom phenol layer was discarded after the final chloroform step, DNA

was precipitated from the top aqueous phase by adding 6ml ofcold (-200 C) 9S% ethanol

and mixing weil. DNA was spooled with a sterile glass micropipette (with a heat-sealed

tip), resuspended in 200-4001l1 ofTris-EDTA (TE, pH 8.0), and stored at 40 C until

further use.

23 b) From paraffin blocks

Sections ofparaffin-embedded tissue stained with hematoxylin and eosin were exarnined under a microscope, and nonnal and neoplastic regions were marked onto corresponding unstained tissue blocks. Before slicing sections from paraffin-embedded tumours, areas ofnormal tissue were dissected away. For normal tissue, only excess

paraffin was removed. Five to six 20llm tissue sections were placed in l.5ml tubes.

Paraffin was removed by two seriai washes with 1ml xylene, followed by two washes in

absolute alcoho!. The samples were air dried for 10 minutes at room temperature. The

tissues were then resuspended in 1m1 digestion buffer containing 50mM Tris (8.3), 1mM

EDTA, 0.5% Tween 20 and 200mg proteinase K, and incubated at 55 0 C ovemight.

Sarnp1es were subsequently heated to 95 0 C for 10 minutes to inactivate the proteinase K.

The tissues were then extracted once with equal volume ofphenol, twice with phenol­

chlorofonn (1: 1), and once with chlorofonn. DNA was precipitated by adding 1/3

volume of 10M anunonium acetate and 2.5 volumes ofethanol to the aqueous layer and

allowing sarnples to sit at -200 C for 15 minutes. DNA pellet was recovered by spinning

at 15,000 rpm for 10 minutes. After washing with 70% ethanol, the DNA was

resuspended in 50111 TE (pH 8.0).

24 2.3 Mutation Screening a) PCR Amplification

Primer pair sequences used for PCR amplification ofCDKN2A exons 1 and 2 and flanking intron regions are listed in Table 2.2. Genomic DNA (100ng) was amplified in

SOJ.l1 reaction mixtures containing IX buffer (SOmM KCl, 10mM Tris-HCI, pH 9.0

Gibco-BRL,USA), 1.0 mM MgCI2, 5% dimethyl sulfoxide (DMSO), 200J.lM dNTPs,

200ng ofeach primer, one ofwhich was biotinylated, and 0.2 U ofTaq DNA Polymerase

(Pharmacia, Sweden). PCR conditions consisted ofan initial denaturation at 950 C for 5 minutes, 35 cycles of 15 seconds at 950 C, 30 seconds at 55-580 C, 30 seconds at noc, followed by a 10 minute extension at n oC in a Perkin-Elmer 9600 Thermocycler

(Perkin-Elmer, USA). Reaction products were electrophoresed on 1% agarose gels containing 1 mg/ml ethidium bromide. Bands corresponding to amplified DNA fragments were sliced from the gel and placed in 1.5 ml tubes with 300ml ofsterile water.

DNA was isolated from the agarose by repeatedly freezing and thawing. Subsequently,

DNA in the supematant was removed from the gel pieces, and 37.51-l13M sodium acetate and 300J.l1 ofcold isopropanol were added. Samples were placed at -200 C to facilitate precipitation ofDNA and then microfuged at 15,000 rpm for 10 minutes. Excess isopropanol and sodium acetate were decanted and the DNA pellet was rinsed with 300J.ll

70% ethanol. Purified PCR product was resuspended in 50J.l1 TE (pH 8.0) for the beads reaction.

25 •

TABLE 2.2 Sequences of primers used for PCR amplification and direct sequencing of CDKN2A exons 1 and 2. Table 2.2 Primer Sequences for CDKN2A Mutation Analysis

Name Sequence Use

p16-1F 5'-GAAAGGAGAGGAGGGGCT-3' Amplify and sequence exon 1 p16-1R 5'-GCGCTACCTGAnCCAATC-3' Amplify exon 1 p16-2F 5'-GGAAAnGGAAACTGGAAGC-3' Amplify exon 2 p16-2R 5'-TCTGAGCTTIGGAAGCTCT-3' Amplify exon 2 p16-S1 5'-GCCnCGGCTGACTGGC-3' Sequence exon 1(-) strand p16-S2 5'-GCGCCACCGCCTCCAGC-3' Sequence exon 1 (+) strand p16-S4 5'-TCAGCCAGGTCCACGGGC-3' Sequence exon 2 (-) strand p16-S5 5'-nCCTGGACACGCTGGTG-3' Sequence exon 2 (+) strand b) Beads Reaction • lnitially, 30111 ofbeads per sample were aliquoted into I.Sml tubes and placed into a magnetic rack (Dynal, Norway) to catch the magnetic beads. Supematant was removed

and the beads were removed from the rack and 'washed' (ie. mixed by gently pipetting up

and down) in 60111 of2M LiCI three times and then resuspended in 20111 LiCI per sample.

To atlach biotinylated double-stranded PCR fragments to the streptavidin-coated beads

(Dynabeads M-280 Streptavidin, Dynal), 20111 ofthe beads :-vere added to each DNA

sample and incubated at 370 C for 30 minutes, mixing every 10 minutes. The beads with

attached DNA were subsequently washed in ISOll12M LiCI twice. Finally, to separate

non-biotinylated PCR product, 40111 of0.2M NaOH was added to each tube, incubated at

room temperature for S minutes and supematant removed. The denaturation step was

repeated and non-biotinylated DNA in supematant was precipated by adding SOIlI of2M • Tris (pH 7-8) and 300111 absolute ethanol. Single-stranded DNA (either in pellet form for the non-biotinylated strand, or on the beads for the biotinylated strand) was resuspended

in 10111 sterile water for sequencing.

c) Direct DNA Sequencing

For sequencing ofsingle-stranded DNA, the dideoxy chain termination method a,

initially described by Sanger et al. 79was employed using T7 sequencing kit (Pharmacia.

Sweden). To anneal sequencing primer to the DNA template, 2111 (200ng) ofprimer

(sequence listed in Table 2.2) and 2111 ofannealing buffer were added to the templatc

26 DNA and incubated at 650 C for 10 minutes. Tubes were aIlowed to cool to room • temperature for approximately 30-45 minutes and subsequently placed on ice. To prepare for the termination reactions, four 0.5ml tubes were labeIIed 'A', 'C',

'G', and 'T' per sample, and 2.51!1 ofdideoxy nucleotide mix was pipetted into the

corresponding tube.

For the labeIIing reàction mixture, 31!1 ofLabeIling Mix 'A' and II!I ofu­

[35S]dATP (Amersham, Sweden) were added to the tubes containing annealed template

and primer, and mixed by gentle pipetting. Subsequently, 21!1 ofT7 polymerase (diluted

Ils with enzyme dilution buffer) was incubated with the template, primer, labeIIing mix

and u-[35S]dATP at room temperature for 5 minutes.

Four I!I ofthe above mixture was transferred into each ofthe pre-warmed tubes

labeIIed 'A', 'C', 'G', and 'T'and incubated at 370 C. After 5 minutes, the dideoxy

termination reaction was stopped by adding SI!l ofStop Solution to each tube. The

samples were heated at 950 C for 10 minutes and then placed on ice. Four I!I ofsample

was loaded onto a 6% polyacrylamide (containing 19:1 acrylamide: bis, IX TBE (Tris­

borate) 8M urea, 441!1 tetramethylethylenediamine (TEMED), 1201!120% ammonium

persulphate (APS), O.4mm thick) gel and run at a constant power of60W for 1-2 hours.

Gels were transferred to chromatography paper (3MMChr, Whatrnan, UK) and vacuum

dried (Model 583 Gel Dryer, BioRad, USA) before exposing to autoradiography film

(Hyperfilm-MP, Amersham, Sweden) for 2-6 days.

27 2.4 Characterisation of mutations • a) Restriction enzyme digestion The Met53Ile alteration was found to create a Sau3AI restriction enzyme site. To

confum the presence ofthis change, 1f.!1 ofSau3AI enzyme (1 Ounits; Phannacia, Sweden)

was added to a 0.5ml tube containing 2f.!1 10X One-Phor Ail Buffer PLUS (Pharmacia), 2

f.!1 sterile water and 15f.!1 exon 2 PCR product, for a total reaction volume of20f.l1 which

was incubated at 370 C for Iwo hours. Digestion products (ranging 40 to 469 basepairs in

size) were electrophoresed using a 2.5% agarose gel containing 1mg/ml ethidium bromide

and 1XTBE.

b) PCR-SSCP analysis

Single-strand conformation polymorphism (SSCP) analysis was performed to

screen cases and controls for the Met53Ile alteration. CDKN2A exon 2 and flanking

introns were PCR-arnplifed using primers listed in Table 2.2. Genomic DNA (100ng)

was arnplified under standard PCR conditions in IX buffer (50mM KCl, 1OmM Tris-HCl,

pH 9.0, Gibco-BRL, USA), 1.0 mM MgCI2, 5% DMSO, 200f.lM dNTPs without A, 25f.!

M dATP only, 1.0f.!1 a-[35S]dATP, and 100ng ofeach primer in a final volume of

twenty-five microlitres. PCR conditions consisted of 1 cycle of5 minutes at 95 0 C, 35

cycles of 15 seconds at 950 C, 30 seconds at 550 C, 30 seconds at noc, followed by a 10

minute extension at n oC in a Perkin-Elmer 9600 Thermocycler. Reaction product was

diluted with 8f.!1 ofdenaturing loading buffer (95% deionized formarnide, 10mM EDTA,

0.05% bromophenol blue, and 0.05% xylene cyanol) and denatured at 950 C for 10

28 minutes before loading ante a 6% polyacrylamide gel containing 29:1 acrylamide:bis,5% glycerol, IX TBE, TEMED and APS. Samples were electrophoresed at a constant power of 1-2W at room temperature ovemight (20-24 hours). Gels were dried and exposed to film as described above.

dl PCR-microsatellite analysis

Primers for dinucleotide microsatellite markers flanking and within the CDKN2A locus used for haplotype analysis offamily 231 are described in Table 2.3. The markers included (from telomere to centromere): D9S161, IFNA, D9S942, D9S126, D9S171,

D9S169. The 25111 PCR mixture contained IX buffer (Pharmacia), 200ilM dNTPs without dATP, 251lM dATP only, 1.0 III u_[35S]dATP, 100ng ofeach primer, O.1U Taq polymerase (Pharmacia) and lOOng genomic DNA. The thermocycling conditions used were the same as those for PCR-SSCP analysis ofCDKN2A exon 2 (section 2.4(b)).

Eight III ofdenaturing loading buffer was added to PCR mixture and heated 10 minutes at

95 0 C. Four III ofeach PCR reaction was loaded ante denaturing 6% polyacrylamide gels

(identical to gels used for sequencing) and electrophoresed at 60W for 1-2 hours. Gels were prepared as described above. AUeles were sized using M13 sequence standard

(Pharmacia) and compared to positive controls (described in Results).

29 TABLE 2.3 Sequences of primers used for CDKN2A haplotype analysis. •

Table 2.3 Primer Sequences for CDKN2A Haplotype Analysis

Name Sequence Locus Heta Size Rangeb

D9S162-F 5'-AATICCCACAACAAATCTCC-3' 9p23-p22 0.72 172-196 D9S162-R 5'-GCAATGACCAGTIAAGGTIC-3' 9p23-p22 IFNA-F 5'-GTAAGGTGGAAACCCCCACT-3' 9p22 0.72 138-150 IFNA-R 5'-TGCGCGTIAAGTIAATIGGTI-3' 9p22 D9S942-F 5'-GCAAGATICCAAACAGTA-3' 9p21 0.95 101-131 D9S942-R 5'-CTCATCCTGCGGAAACCATI-3' 9p21 D9S171-F 5'-AGCTAAGTGAACCTCATCTCTGTCT-3' 9p21 0.79 159-177 D9S171-R 5'-ACCCTAGCACTGATGGTATAGTCT-3' 9p21 D9S126-F 5'-CAACTCCTCTIGGGAACTGC-3' 9p21 0.68 238-248 D9S126-R 5'-ATIGAAACTCTGCTGAATITICTG-3' 9p21 D9S169-F 5'-AGAGACAGATCCAGATCCCA-3' 9p21 0.82 259-274 D9S169-R 5'-TAACAACTCACTGATIATTIAAGG-3' 9p21

ahet=heterozygosity; ·size range in base pairs Chapter 3 • RESULTS

3.1 Mutation analysis

The results from sequence analysis ofCDX.V2A exons 1 and 2 are summarized in

Table 3.1. Index cases from seventy-five kindreds were screened for CDKN2A mutations

and one family (231) was found to harbour a gerrnline G>C nuc1eotide change at position

159 in exon 2, corresponding to a methionine to isoleucine amino aeid substitition at

codon 53 (Figure 3.1 a). The heterozygous Met53Ile alteration was detected in a woman

who developed two cutaneous malignant melanomas at age 40. The presence ofthe

mutation was confinned by sequencing constitutional DNA twice, extracted from two • separate blood samples drawn from the index case (individuaI231-1 in Figure 3.2). Subsequently, sequencing and SSCP analysis ofblood DNA from her unaffected 89 year

old maternai aunt (231-2) revealed that she is a carrier ofthe Met53Ile gennline mutation

as weil. A detailed family history revealed that no other members ofthe family have had

melanomas, although the index case's son, who is now 16 years old, had a large multiple

compound nevus removed from his mid-scapular region in childhood. The index case's

mother, an obligate carrier of the mutation, was diagnosed with multiple primaries: she

developed intraductal carcinoma of the breast at 73, numerous villus polyps of the

colon inc1uding one with a carcinoma in situ at77, and died of mouth cancer at 79.

Since her mother was deceased, archivai tumour tissue was obtained with the daughter's

consent in order to analyse nonnal and tumour tissue. According to Knudson's 'two-

30 5 hit' model for tumour suppressor genes , if the inheritance of the Met53lIe alteration • predisposes to cancers in this family, analysis of these tumours should show retention of the mutant allele accompanied by a somatic event that results in the loss of the wild­

type allele during tumourigenesis. Unfortunately, despite several attempts, peR

amplification of DNA extracted from paraffin-embedded tissue was unsuccessfuI.

Although it is not known how these tissues were preserved, the poor quality of DNA

may have been attributable to the initial fixation method, as sorne are methods are more

82 efficient for amplification of DNA than others • Given that the tumour samples were

over ten years old, it was also likely that the DNA had degraded. Thus, loss of

heterozygosity (LüH) analysis of archivai tissue was Iimited by difficulties in obtaining

intact DNA. The development of a mutation-specifie antibody to CDKN2A in the

future would circumvent these difficulties.

In addition, the index case's maternai uncle was diagnosed with bladder cancer al

age 71, the maternaI grandmother was possibly diagnosed with gall bladder cancer at 62.

and the great-grandmother had oral cancer in her 50s. These relatives are deceased and

archivaI tissue was unavailable for analysis. Furthermore, the index case's brother was

contacted and declined to participate in this study. The index case's children, ail und~r 11\

years old, were too young to consent to genetic testing.

A previously described variant in the CDKN2A gene was also detected in 5

families in this study (299, 306, 385, 207, 296). A G>A transition at nucleotide 442 in

exon 2 results in a conserved amino acid change from alanine to threonine at codon 148

(Figures 3.3a & b). The Alal48Thr variant appears to be a neutral CDKN2A

31 polymorphism; it has been reported in the germline ofa total of23/357 individuals from

6 74 65 66 affected and control populations in the USA !.62, Sweden , and Australia • and recent functional analysis has shown that this variant behaves similarly to wild-type

77 78 CDKN2A • • The frequency ofthe Ala148Thr polymorphism in this study of75

Canadian families is consistent with other reports (6-7% ofthe general population). No other CDKN2A alterations were detected in the remaining sixty-nine families.

To supplement the CDKN2A mutation analysis data, 35 families were inc1uded in separate studies looking for germline alterations in other genes implicated in hereditary cancer, including TP53, BRCAl and/or BRCA2. With the exception offamilies 214 and

284 where BRCAl and BRCA2 alterations, respectively, have since been found (P.Tonin, personal communication), no mutations have been reported so far (Table 3.1). Thus, it

appears that cancer susceptibility in these families is Ilot attributable to mutations in these

genes.

3.2 Restriction enzyme digestion

The G>C base change producing the Met53Ile variant in family 231 was found to

create a Sau3AI restriction site, from GATQ to 'GATe.. PCR amplification and

subsequent restriction enzyme digestion ofexon 2 fragment (509 bp) from wild-typc

DNA yielded two fragments 469 and 40 bp in size. Sau3AI digestion product ofDNA

heterozygous for the Met53Ile change produced an additional two bands, 314 and 155bp

in size (data not shown).

32 •

TABLE 3.1 Summary of results from mutation analysis of seventy-five cancer families (Group 1: Multiple primaries in index case; Group 2: Melanoma in index and multiple cancers in family; Group 3: Other cancer in index case and numerous cancers in the family; Group 4: No cancer in the index with many cancers in family). For pedigrees see Appendix A. Table 3.1 Summary of Mutation Analysis Results

• Group No. FamilyNo. CDKN2A Other genes analysedb

112 TP53 161 BRCA1, TP53 210· 214 TP53, BRCM- 221 TP53, BRCAlIZ·M 231· Met5311e TP53 233 TP53 239 TP53, BRCMdM 242 TP53 299 Ala148Thr 306 Ala148Thr 320 323 324 TP53 329 333 365 385 Ala148Thr 386 TP53 438 445 449 BRCA2 479 TP53 647 BRCA2

2 157 TP53 191 192 194 TP53 196 197 203 204 207 Ala148Thr 210 223 228 231 Met5311e TP53 240 BRCM 249 250 284 BRCA1, BRCA2- 295 TP53 552 M 607 BRCA11Z N2605 N2621 N2796 Pagination Error Erreur de pagination Text complete Le texte est complet

National Library of canada Bibliothèque nationale du Canada Canadian Theses Service Service des thèses canadiennes • Table 3.1 (cont'd) Group No. Family No. CDKN2A Other genes analysedb

3 108 BRCA2, TP53 114 TP53 117 TP53 122 TP53 124 TP53 126 TP53 134 135 138 BRCA2 144 148 TP53 152 TP53 154 173 TP53 195 BRCA1 208 TP53 224 TP53, BRCA lIt'M 259 BRCM 265 281 296 Ala148Thr 301 303 305 402 TP53 481 559 756

4 199 466

a in Group 2 as weil bin separate studies, a subset of families were screened for mutations in other genes with negative results, unless indicated by (*): BRCM analysis: Simard et a/., 199480 BRCA2 analysis: Phelan et a/., 199681 BRCA11t'M analysis of founder mutations seen in Ashkenazi Jewish population in BRCM (185deIAG, 5382insC, 188de111) and BRCA2 (6174delT): Tonin,P. (unpublished data) TP53 analysis: Malkin, D. (personal communication)

32 FIGURE 3.1 a) Direct sequencing results on PCR amplified CDKN2A exon 2. Identification of the Met5311e mutation in individuals 231­ 1 and 231-2 (designated mutant) are shown next to control sequence (wild-type).

FIGURE 3.1b) Single-stranded conformation polymorphism (SSCP) analysis of CDKN2A exon 2. Variant bands corresponding to the Met5311e mutation are indicated by (M) and shown with the wild-type pattern (W). •

a Met5311e (G159~C)

wild-type mutant mutant 5' A CGT ACGT ACGT 5' ~ ~ ~ ... -:-:- -. A"-_ .... .-- .. --_ JA ~r _- ~ .., __ T ...... _ .. ------~ il3----.­ -____r_"GL& :. at--=: JEI._ . - 3'

• b M WWWW MM

.. 1 Cts • CC»'" FIGURE 3.2 Pedigree structure of family 231. Individual numbers (231-1 and 231-2) are shown above and 10 the left of pedigree symbols. Abbrevialions are: ca: cancer d: age al death dx: age al diagnosis PSU: primary site unknown yrs: age in years •

Family 231

PSU 50+ oral ca

breast ca-dx73 bladder ca 89yrs colon ca-dx77 dx71 , d77 mouth ca-dx79 FIGURE 3.3a) Direct sequencing results on PCR amplified CDKN2A exon 2. Identification of the Ala148Thr polymorphism (mutant) shown next to control sequence (wild-type).

FIGURE 3.3b) Detection of the Ala148Thr polymorphism by direct sequencing using the 'mutation loading' method (ie. loading 'A's together, 'C's together, etc). The CDKN2A variants seen in index cases from families 299 and 306 are indicated byarrows. • a Ala148Thr (G442~A)

wild-type ACGT

rt1ii~ 3' ... •...... /G ,==-\~ \ -~-- 5' ;;---­

b

C G T 3.3 SSCP analysis

The MetS3Ile alteration was also confirmed as a detectable shift by single­ stranded confonnation polymorphism (SSCP) analysis ofthe S09bp CDKN2A exon 2

PCR product, which was used as the most effective method ofscreening for the variant

(Figure 3.1 b). No altered SSCP migration patterns corresponding to this change were detected in any ofthe other 74 individuals in this study, nor in SO controls from Montreal ofmulti-ethnic background (40% French Canadian; 20% English Canadian; IS%

Ashkenazi Jewish; 2S% other) who did not have cancer.

The MetS3Ile mutation was previously reported in two Australian melanoma kindreds65 and it has since been detected in a third Australian family (Walker, personal communication). The three families from Austra1ia are ofScottish descent and interestingly, family 231 was also found to originate from the west ofScotland.

Consequently, constitutional DNA from 120 Scottish controls were screened by SSCP analysis using family 231 as a positive control and no samples were found to have the

Met53Ile variant. Ninety-seven ofthese were Scottish women who were referred to the

Department ofMedical Genetics at the University ofAberdeen for predictive testing for hereditary diseases other than cancer, inc1uding myotonic dystrophy, Huntington's and fragile-X (B. Miiner, personal communication). The remaining people screened were women diagnosed with breast cancer ascertained at the Beatson Institute, Glasgow,

Scotland (D. Black, personal communication). In total, the Met53Ile was not seen upon

analysis of438 chromosomes for this variant in Scottish and Canadian populations.

33 3.4 Haplotype Analysis

To address the possibility that farnily 231 and the other Australian kindreds with the Met53I1e alteration may share a common ancestor, we obtained blood DNA from 5

Met53I1e carriers affected with melanoma from Iwo ofthe Australian families (60001 and

41001) to use as positive controls for haplotyping farnily 231. The results from peR­ microsatellite analysis ofthese three families using six 9p21 markers flanking the

CDKN2A gene are shown in Figure 3.5 and combined with data from mutation analyses in Figure 3.6. The marker D98942 is close to CDKN2A, <30kb proximal to the gene and is highly polymorphie (heterozygosity =0.95). D98162 and IFNA lie approximate1y 1­

2Mb and 500kb telomeric to CDKN2A, respectively. D9S171 and D9S126 are located about 1.5Mb and 2Mb centromeric to the CDKN2A gene. As initially reported by Walkcr

6S et a1. , the Iwo 9p-linked Australian melanoma kindreds 60001 and 41001 are apparently unrelated, having Iwo different haplotypes that cosegregate with the Met53I1e mutation.

In particular, these haplotypes in these Iwo Australian kindreds differ at the D9S942 and

D98126 loci. Since the D9S942 marker itself is located less than 30kb proximal to

CDKN2A, il is unIikely that families 60001 and 41001 have a common ancestor.

Interestingly, however, genotyping offamily 231 has revealed that individuals from this kindred and family 41001 share identical alleles for the markers

D9816211FNAlD9S9421D9SJ711 D98126 (174/148/119/165/242/267) which represent

the segregating haplotype in the latter family. Preliminary haplotyping ofthe third

Australian farnily with this mutation has suggested that il is related to family 41001 as

weil (N. Hayward, personal communication). Thus, it appears that these three families

34 with the Met53Ile mutation share a common founder and the mutation has probably arisen

a second time in family 61001.

•

35 •

FIGURE 3.4 Results from haplotype analysis of individuals from families carrying the Met5311e mutation. The six markers analysed were (from telomere to centromere): 098162,IFNA, 098942,098171,098171,098126 and 098169. Sample numbers at top correspond to individuals from families 41001,60001 and 231. The common allele sizes shared by • families 41001 and 231 are indicated by arrows on the left. The COKN2A gene lies between the 098942 and IFNA loci; 098942 is less than 30kb proximal to COKN2A. AIIele Size (bp)

174 -1 095162

148 ~ IFNA

}CDKN2A

095942

095171 165 --l

242 ~ 095126

267 ~ 095169 e.

Figure 3.5 Pedigrees offamilies 231, 41001 and 60001 incorporating results of haplotype and COKN2A mutational analyses. Identication numbers for the individuals analysed are shown above and to the left of the pedigree symbol. A (+) indicates the presence of the Met5311e mutation, whereas a (-) represents no mutation found. The haplotypes are shown below the pedigree symbol corresponding to allele sizes using the following markers (from top to bottom): 09S162, IFNA, 09S942, 09S171, 09S126. Haplotypes segregating with the melanoma in families 41001 and 60001, and shared by kindred 41001 and 231 are boxed in. Recombination events are indicated by (x). • Chapter4 • DISCUSSION

The CDKN2A gene has been posed as a strong candidate for the melanoma

susceptibility locus, MLM2, on chromosome 9p21; somatic mutations or deletions ofthis

gene are commonly found in melanomas and melanoma-derived celllines and several

CDKN2A germline mutations are seen in familial atypical multiple mole melanoma

(FAMMM) kindreds. Early epidemiological studies have suggested an association

between FAMMM and increased risk ofother cancers, particularily gastrointestinal

83 84 cancers • . Recent reports have also demonstrated CDKN2A is frequently inactivated in

the development ofseveral types ofcancers, indicating its universal role in

tumourigenesis. The relationship between CDKN2A germline mutations and the risk of

cancer is still unclear. To determine whether or not CDKN2A plays a role in a more

general group ofcancer families or whether in fact CDKN2A germline mutations are

restricted to FAMMM, 75 index cases from cancer-prone kindreds that do not necessarily

fit the pattern ofa hereditary melanoma syndrome were analysed for predisposing

CDKN2A mutations.

Met53l1e: mutation or polymorphism?

A CDKN2A germline alteration was detected in one family (231). Th", missensc

mutation results in a methionine to isoleucine substitution at codon 53 and falls outside of

ahighly-conscrved region ofthe ankyrin repeats ofthe pl6CDKN2A protcin. Il is also

36 interesting to note that the related S. cerevisiae protein, Ph081p, contains an isoleucine • instead ofa methionine at the corresponding position (Figure 4.1), indicating that a Met53I1e substitution in other species is non-deleterious. Furthermore, the Met53I1e

germline alteration was detected in a woman from family 231 who is healthy at 89 years

old. Given these observations, it is possible that this change simply represents a neutral

variant ofCDKN2A.

On the other hand, evidence suggesting that Met53I1e may be a deleterious

mutation exists: firstly, an amino substitution replacing a methionine with an isoleucine

is a non-conservative change that can potentially interfere with normal pl6CDKN2A

protein structure and function. In particular, methionine residues are frequently present in

a-helical secondary structures due to their size. In contrast, the p-carbon in isoleucine

tends to destabilize the a-helix because ofsteric hindrance and this residue is found more

often in a p-sheet conformation85 (Figure 4.2). The absence ofan electronegative sulphur

atom in isoleucine may affect the folding and function ofthe pl6CDKN2A protein.

Secondly, although the Met53I1e substitution occurs outside ofthe ankyrin

consensus sequence, a methionine is found at this site in four offive known human

CDKN2A proteins (p14, pIS, pl6 and p19), indicating sorne lever ofconservation ofthis

amino acid in related human proteins (Figure 4.1). Random mutagenization ofthe

CDKN2A gene has demonstrated that a Met53Thr mutation affects the binding activity of

86 pl6CDKN2A to CDK4 • Thus, it appears that the methionine residue at position 53 is

important for the biological activity ofthe protein.

37 •

FIGURE 4.1 Alignment of human CDKN2 cell cycle inhibitors with Ph081 p kinase inhibitor from S. cerevisiae. Ali of the proteins shown contain 4 ankyrin-like repeats with high similarity in repeats )-IV. Conserved amine acid residues belonging to the ankyrin consensus sequence are in bold lellers. An arrow indicates the location of methionine 53. • ankyrin repeat 1 11 43 p14 GSDEGLASAAARGLVEKVRQLLEAF-ADPNGVNR p15 GSDEGLA-TPARGLVEKVRHSWEAG-ADPNGVNR p18 PWGNELASAAARGDLEQLTSLLQNN-VNVNAQNG p19 VRAGTLSGAAARGDVQEVRRLLHRELVHPDHLNR p16 PSADWLATAAARGRVEEVRALLEAG-ALPNAPNS Pho81p VQFDPLNVACKFNNHDAAKLLLEI-RSKQNADNA ankyrin repeat Il 44 76 p14 FGRRAIQVMMMGSARVAELLLLHGAEPNCADPA p15 FGRRAIQVMMMGSARVAELLLLHGAEPNCADPA p18 FGRTALQVMKLGNPEIARRLLLRGANPDLKDRT p19 FGKTALQVMMFGSTAIALELLKQGASPNVQDTS p16 YGRRPIQVMMMGSARVAELLLLHGAEPNCADPA Pho81p LCTLHIVAKIGGDPQLIQLLIRYGADPNEIDGF t ankyrin repeat III 77 109 p14 TLTRPVHDAAREGFLDTLVVLHRAGARLDVRDA p15 TLTRPVHDAAREGFLDTLVVLHRAGARLDVRDA p18 GFA-VIHDAARAGFLDTLQTLLEFQADVNIEDN p19 GTS-PVHDAARTGHLDTLKVLVEHGADVNVPDG p16 TLTRPVHDAAREGFLDTLVVLHRAGARLDVRDA Pho81p NKWTPIFYAVRSGHSEVITELLKHNARLDIEDD ankyrin repeat IV 110 142 p14 WGRLPVDLAEERGHRDVAGYLRTATGD-----­ p15 WGRLPVDLAEERGHRDVAGYLRTATGD-----­ p18 EGNLPLHLAAKEGHLRVVEFLVKHTASNVGHRN p19 TGALPIHLAVQEGHTAVVSFLA--AESDLHRRD p16 WGRLPVDLAEELGHRDVARYLRAAAGGTRGSNH Pho81p NGHSPLFYALWESHVDVLNALLQRPLNLPSAPL FIGURE 4.2 Structure of methionine and isoleucine. •

COO· COO· 1 1 +H3N-C-H +H3N- C-H 1 1 CH2 H3C-PC-H 1 1 CH 2 CH2 1 1 S CH 3 1 CH 3

Methionine Isoleucine (Met, M) (Ile, 1)

Figure 4.2 Thirdly, the Met5311e alteration cosegregates \Vith melanoma susceptiblility in • three large Australian kindreds65 (G. Walker, personal communication), consistent \Vith the possibility that this missense mutation may be predisposing to melanoma in these

families.

Additional data supporting the postulate that Met5311e is a disease-related

mutation has come from screening populations for the CDKN2A variant. ln this study, a

total of438 chromosomes from Scottish and Canadian controls \Vere analysed for the

putative mutation \Vith negative results. ln addition, none of72 unaffected controls

screened by allele-specific oligo (ASO)hybridisation by Walker et al. \Vere found to have

65 this alteration • These results make it unlikely that Met5311e is a neutral polymorphism;

rather, it appears to be a bonafide mutation. However, confirmation ofthe biological

significance ofthis allelic variant ofCDKN2A awaits 1) functional experiments studying

the CDK4/6 binding and cell cycle inhibitory abilities ofthe mutant pl6CDKN2A and 2)

examination ofturnours ofmutation carriers for the retention ofthe Met5311e allele and

the loss ofthe wild-type CDKN2A allele by a second somatie event.

CDKN2A mutations in non-FAMMM kindreds

The putative CDKN2A mutation was detected in a non-FAMMM kindred with one

case ofmelanoma and numerous cancers elsewhere. An obligate carrier ofthe Met5311e

change in family 231 developed multiple primaries ofthe breast, colon and mouth in her

70s. There were also cases ofbladder and oral cancers, as well as a suspected gall bladder

cancer in other relatives. These findings are intriguing in light ofevidence supporting the

38 involvement ofCDKN2A in sporadic cancers ofthese types. It has been observed that 60­

41 62 65% ofprimary breast tumours and celllines show homozygous deletion ofCDKN2A •

44 and the gene is inactivated by 5'CpG methylation in 33% ofbreast cancer celllines •

Ninety-two percent ofcelllines derived from colon primaries demonstrate de novo methylation ofCDKN2A as well. Moreover, frequent CDKN2A mutations have been reported in oral squamous cell carcinomas and biliary tract cancers 45. Therefore, it is tempting to speculate that the Met53Ile mutation may predispose to other types of malignancies in this family. Unfortunately, the absence ofsufficient tumour tissue in these affected individuals precludes CDKN2A loss ofheterozygosity (LOH) analysis and this hypothesis cannot be tested.

The majority ofgermline CDKN2A mutations identified to date occur in FAMMM kindreds. Recently, however, there have been other recent reports ofgermline CDKN2A mutations in families that do not fit the pattern ofan hereditary melanoma syndrome.

Whelan et al. identified a GlylOITrp CDKN2A mutation in a family with one case of melanoma and pancreatic cancer, two cases ofpancreatic cancer only, and one occurrence

72 ofsquamous cell carcinoma ofthe tongue • In addition, Yarbrough et al. have detected an in-frame CDKN2A germline deletion removing amine acids 96-99 in a family with melanoma, non-small celllung cancer and squamous cell carcinoma ofthe head and

70 neck . Thus, CDKN2A germline mutations are not restricted to FAMMM.

39 Other cancers in FAMMM kindreds • FAMMM kindreds contain CDKN2A mutation carriers who develop other cancers. In particular, molecular epidemiological studies have provided evidence that

sorne, but not ail, FAMMM families with CDKN2A mutations are more likely to develop

64 87 pancreatic cancer • • The reported association may be attributed, in part. to

ascertainment bias: an investigation ofAustralian melanoma kindreds with CDKN2A

mutations found no significant excess ofpancreatic cancer in these families (N. Hayward.

personal communication). It is also possible that the position or type ofthe mutation

within the CDKN2A gene is related to the type ofcancer risk: in hereditary breast cancer,

mutations in the 3' third ofthe BRCAl gene are associated with a significat:iiy lower

88 proportion ofovarian cancer to breast cancer cases in BRCAl-linked families • and in

von Hippel Lindau disease kindreds, risk ofphaeochromocytoma is greatest in thosc with

89 • missense mutations in the VHL gene • Additional genetic and environmental factors may contribute to risk ofpancreatic cancer in melanoma-prone families and explain the

conflicting data.

Malignancies other than melanoma and pancreatic carcinoma reported in

CDKN2A mutation carriers in FAMMM families include one case ofan adenocarcinoma

9D 73 ofunspecified type , a smoking-related laryngeal carcinoma • an early onset brcast

62 74 cancer and prostate cancer , and cervical, breast cancer and non-Hodgkin's lymphoma •

Furthermore, several second primaries including bowel cancer, multiple myeloma,

squamous ceU carcinoma ofthe mandible, squamous ceU carcinoma ofthe vocal chord,

and two cases oflung cancer, have been observed in Australian melanoma families (N.

40 Hayward, personal communication). These data provide additional evidence suggesting that individuals who inherit CDKN2A mutations are not only predisposed to developing melanoma, but are at increased risk ofdeveloping other cancers as weil, particularly pancreatic cancer and squamous cell carcinoma ofthe head and neck.

Penetrance of CDKN2A mutations

The Met53Ile mutation has also been identified in three Australian families with numerous cases ofmelanoma but no excess ofother cancers in mutation carriers (G.

Walker, personal communication). The high incidence ofmelanoma in the Australian kindreds but not in the Canadian family may be due to an interaction ofthe CDKN2A mutation with environmental factors such as the high UV flux over Australia. Evidence supporting the role ofUV radiation in modulating the phenotypic penetrance ofCDKN2A mutations has been reported: in an Australian study of 151 putative mutation carriers in

18 melanoma families, individuals bom after 1959 had a cumulative incidence 21-fold greater than those bom after 1959, suggesting that the penetrance ofCDKN2A mutations may be increasing as a result ofincreased sun exposure to carriers due to lifestyle

91 changes • ln keeping with these observations, in family 231, the two CDKN2A mutation carriers who did not deve10p melanoma were also the oldest individuals, both bom before

1959.

The occurence ofa healthy 89 year old mutation carrier in family 231 is consistent

65 with previous reports ofunaffected Met53Ile mutation carriers in other farnilies • The penetrance for the development ofmelanoma in CDKN2A mutation carriers arnong

41 61 62 62 6S different families from North America • , Europe and Australia ranges from 69-78%. • Thus, from an individual perspective, the probability ofdeveloping melanoll''1 :n a person with a CDKN2A mutation is high, although not 100%.

Curiously, there are no reported mutations in families with multiple cancers that

do not include melanoma. Although ascertainment bias from other studies make it

difficult to evaluate actual risk, the results from this study suggest that from a familial

perspective, the likelihood ofdeveloping melanoma in a family with a CDKN2A mutation

appears to be very high. Thus, CDKN2A is highly penetrant for cutaneous malignant

melanoma, apparently more so than BRCAl is for breast cancer; mutations in the breast

and ovarian cancer susceptibility gene, BRCAl. have been detected in severallarge

families that inc1ude numerous cases ofovarian, without any cases ofbreast cancer (P.

Tonin, personal communication). A biological explanation may be that melanocytes ma)'

lack functionally redundant genes that enable other cell types to tolerate CDKN2A

mutations.

Significance of negative results

No other disease-related CDKN2A mutations were found in the other seventy-four

families in this study. The a priori expectation was that a relatively small number of

CDKN2A germline mutations might be present in this series and therefore no mutations

could afford to be missed. Direct DNA sequencing is a highly sensitive method of

detecting sequence changes, particularly compared to other methods such as single-strand

conformation polymorphism (SSCP) analysis, where the sensitivity ofmutation detection

42 is dependent on many unpredictable factors, including the sequence and size ofthe DNA

92 fragment being analysed, temperature, and gel composition • To this end, the mutation screening strategy in this study involved PCR amplification and direct sequencing of

CDKN2A exons 1,2 and flanking splice junctions. The possibility exists, however, that in sorne families CDKN2A inactivating mutations may have been missed. In particular,

CDKN2A exons 1~ and 3 were not analysed, hence mutations in these exons would not have been identified. The alternative ~-transcript that includes exon 1pdoes not appear to be translated in vivo40 and sequence analysis ofthe newly discovered exon 1pin several

9p-linked melanoma families without CDKN2A mutations and various tumour samples

93 failed to detect any mutations • Given these negative results, exon 1~ was excluded from this study although its significance is unclear. Exon 3 is only Ilbp in size and a1though sequence polymorphisms have been reported in the 3'UTR region ofthe gene, no

45 6 deleterious mutations have been detected so far .4 ; hence, exon 3 was not sequenced. ln addition, heterozygous germline deletion ofan exon or the entire gene would not have been detected, as only the normal allele would amplify in a PCR-based assay. However,

62 no heterozygous germline deletions ofCDKN2A have been reported so far .

A more Iikely explanation for the paucity ofCDKN2A mutations from this analysis is that other genetic and environmental risk factors contribute to risk ofcancer in these pedigrees. Thirty-five ofthe families included in this study were a1so analysed for

mutations in other genes implicated in hereditary cancer, including TP53, BRCAl and

BRCA2 (Table 3.lin Results). Interestingly, a BRCA2 mutation has recently been

identified in family 284 which has a history ofexcessive cancers, including melanoma

43 (p.Tonin, personal communication). A second family (214) has been found to harbour n

BRCAl mutation (ArgI443Ter) in exon 13 (P.Tonin, personnl communication). The proband had multiple primaries including early onset bilateral brenst cancer, pnpillary thyroid cancer and lung cancer.

It is interesting to note that testing ofthe only family in this study that fit the pattern ofa hereditary melanoma syndrome (552; with three affected fi,·~,.degree relatives) failed to detect any CDKN2A mutations. This finding is consistent with the observations that not nli melanoma kindreds are linked to 9p21 and that npproximately only hnlf ofthose kindreds demonstrating linkage to 9p21 have been found to have

CDKN2A mutations6!.62.63.65. In the remaining melanoma families, it is possible that

CDKN2A is inactivated either by mutations in non-coding regions ofthe gene or by germline mutations affecting the methylation pattern ofCDKN2A, hence silencing transcription ofone allele. Further mutation analysis ofnon-coding regions ofthe

CDKN2A gene and epigenetic studies would help test this hypothesis.

Alternatively, other genes may be responsible for melanoma susceptibility. One strong candidate is the CDKN2B gene which lies adjacent to CDKN2A and encodes pI5cDKN2B, another CDK inhibitor that.\s highly homologous to pl6CDKN2A as these two

proteins share a region of81 amino acids with 97% identity34. However, no CDKN2B

germline mutations have been reported thus far. Other cell cycle genes, particularly those

involved in G1 to S phase (restriction point) progression, are attractive candidates for

melanoma predisposition genes. Recently, a CDK4 (cyclin-dependent kinase 4) germline

17 mutation (R24C) was identified in two North American melanoma families • In the

44 normal cell, CDK4 associates with the D-type cyclins and in the absence ofCDK • inhibitors, the cyclin D/CDK4 complexes drive cells through the restriction checkpoint, commiting to another round ofceIl division. Mutant CDK4, however, was found to

demonstrate dramatically impaired binding to either plé DKN2A or plSCDKN2B, although its DKNIB p21CDKNIA or p2'f binding abilities were unaffected. Therefore, melanomas may

have arisen in individuals with the R24C mutation as a result ofCDK4 kinase activity

being less sensitive to inhibition or regulation by specifically by pl6CDKN2A and/or

pISCDKN2B. Thus, in sorne families, inherited CDK4 mutations may confer increased risk

ofmelanoma. A better understanding ofthe genetic risk factors contributing to cancer in

the families with no CDKN2A mutations from this study awaits future identification and

screening ofother cancer susceptibility genes, such as CDKN2B and CDK4.

Ofthe seventy-five families, 24 index cases were c1assified as having had multiple

primaries and ofthese 24 individuaIs, II had developed at least one melaaoma primary.

The index case found to harbour a CDKN2A mutation belonged to the latter subgroup,

having developed double melanoma primaries. The only other index case who developed

multiple primaries ofmelanoma only (from family 210) tested negative for CDKN2A

mutations. Therefore, it appears that CDKN2A germline mutations are most likely to be

found individuals with multiple primaries, including melanoma.

The finding that three offour families with the MetS3Ile mutation appear to share

a common founder is intriguing. The possible existence ofa CDKN2A mutation founder

effect ofScottish origin will facilitate future identification offamilies with the same

mutation. At present, screening ofScottish families with melanoma and other cancers is

45 underway in the west of Scotland (D. Black, Beatson Institute, Glasgow, Scotland). • These families also provide the opportunity to study disease penetrance and other genetic or environmental risk factors on a common susceptibility background.

In addition, the occurence ofthe Met53Ile mutation in an apparently unrelated

family with a different haplotype suggests that a 'mutational hotspot' may exist within the

CDKN2A gene. Given that the Met53Ile mutation has arisen at least twice in the

germline, it is interesting to note that no mutations ofthis codon have been reported in

any turnours or celllines, even though somatic mutations in CDKN2A occur in at least

70% ofthe possible 156 codons making up this small gene (see Tables 4.1 and 4.2 for

compilation ofCDKN2A somatic mutations). However, only 11% ofgermline mutations

identified to date (Figure 1.3 in Introduction) have been seen in primary tumours or ccli

lines. Moreover, ofthe 14 nucleotide positions mutated numerous times in a wide varicty

ofhuman cancers, only 2 are found to be mutated in the germline. There are severai

possible explanations for the lack ofconcordance between somatic and germline

mutations: 1) ascertainment bias: novel germline mutations may be found in kindreds

displaying different phenotypes than the families tested so far, 2) deieterious mutations:

certain CDKN2A germline mutations may not be compatible with life, 3) somatic

mutations reflect mutagenesis by other carcinogens such as UV radiation which induces

characteristic transitions and tandem base changes, and 4) this discrepancy is due to

chance. Further identification ofadditional CDKN2A germline mutations will facilitate

interpretation ofthese data.

46 TABLE 4.1 CDKN2A somatie mutations identified to date (JuJy 1996) in cell Iines. Underlined mutations appear in both cell Iines and primary tumours. Information not specified in the original sources is indicated by (?). Abbreviations include:

paner: pancreas adeno: adenocareinoma SCC: squamous cell earcinoma NSCLC: non-small cell Jung cancer NPC: nasopharyngeal carcinoma HNSCC: head and neck squamous cell careinoma (HNC: head and neck cdll.::er) T-ALL: T-Iymphocyte acute Jymphoblastic leukaemia • Table 4.1: CDKN2A somatie mutations-eelllines BASE EVENT/CODON CELL LINE REF

EXON 1 25 insTG 9 PANCRADENO 94 35C>A Ser12STOP NSCLC 95 55 ins CGCGCAC 19 DUCTAL PANCR 96 58 Ins ACGGCC 20 PANCRADENO 45 58G>C Ala20Pro MELANOMA, LUNG 45 63 dei 23 bp 21 L1VER 45 ? ins 1 bp 23 COLON 45 85 del18 bp 29 PANCRADENO 94 101 dei CGG 34 DUCTAL PANCR 96 104 G>A Gly35Glu MELANOMA 45 106 G>A Ala36Thr PANCRADENO 94 128 dei GT 43 MELANOMA 45 131 dei 33 bp 44 LUNG MUCOEPIDERMOID 45 134 dei G 45 NSCLC SCC 45 142 CC>TT Pro48Leu MELANOMA 45 143 C>T Pro48Leu MELANOMA 45 148 C>T Gln50STOP MELANOMA 45

inll -2 A>C splice CHONDROSARCOMA,NPC 45 inll -2 A>G splice MESOTHELIOMA 45 inll-l G>T splice HNSCC 45 • inll-1 G>A splice NPC 45

EXON2 155T>A Mel52Lys DUCTAL PANCR 96 156 G>C Mel5211e BREAST 97 161 del14 bp 54 ORALSCC 45 167 ins 1 bp 56 MELANOMA 45 171 C>A Ala57Ala MELANOMA 45 171 CC>TT Arg58STOP MELANOMA 45 172 C>T Aro58STOP BLADDER, MELANOMA 45 172 dei 8 bp 58 MELANOMA 45 174 del16 bp 58 ORALSCC 45 180 dei 29 bp 60 T-ALL 98 180 ins 1 bp 60 MELANOMA 45 181 G>T Glu61STOP MELANOMA 45 182A>G Glu61Gly PANCRADENO 94 183 G>C Glu61Asp ORALSCC 99 184 C>G Leu62Vai ORALSCC 99 199 ins 1 bp 67 MELANOMA 45 204 dei 5 bp 68 THYROID 100 205 G>T Glu69STOP MELANOMA, NSCLC 45,95 206 A>T Glu69Vai NSCLC SCC 45 207 G>C Glu69Asp ORALSCC 99 208 C>G Pro70Ala ORAL SCC 99 216 C>A Cys72STOP NSCLC 95 220 G>A Asp74Asn BLADDER 45 231 dei TCTC 77 ORALSCC 45 233# dei TC l§ PANCR ADENO, BLADDER, ORAL 45,46 SCC • 237CC>TI Arg80STOP MELANOMA, L1POSARCOMA 45,101 238 C>T Arg80STOP MELANOMA, MYELOID 45,99, LEUKAEMIA, T-ALL, THYROID, 100,10 ORALSCC 2 242 C>T Pr081Leu MELANOMA 45 247 C>T His83Tyr MELANOMA, NSCLC 45,103 250 G>T Asp84Tyr PROSTATE 45 261 GG>AA Glu88Lys MELANOMA 45 262 G>A Glu88Lys MELANOMA 45 262 G>T Glu88STOP MELANOMA 45 264GG>AA Gly89Ser MELANOMA 45 290 5bp dei 97 MELANOMA 45 295 3bp dei 99 MELANOMA -15 296 GG>CA Arg99Pro MELANOMA 45 ?G>A 101 LEUKEMIA 104 320 G>A Arg107His LEUKEMIA 104 322G>C Asp108His BLADDER 45 329 G>A Trp110STOP MELANOMA 45 330 G>A Trp110srop MELANOMA,OVARY 45 335 G>C Argl12Pro MELANOMA 45 341 C>T Prol14Leu MELANOMA, FIBROSARCOMA 45 346 G>T Aspl16Tyr MELANOMA 101 358 G>T Glu120STOP ORALSCC 99 ? dei 122 LEUKEMIA 104 ?G>C 122 LEUKEMIA 104 369 T>A His123Gin NSCLC 95 • 378 C>T Val126Vai MELANOMA 45,105 386 A>G Tyr129Cys PANCRADENO 94 425A>G His142Arg MELANOMA 45

int2 +1 G>T splice NSCLC 45 inl2 +2 T>C splice DUCTAL PANCR 106

# Because of the ambiguity involved in assigning nucleotide positions to certain deletions, this delelion is numbered from where the wild-type sequence first changes. TABLE 4.2 CDKN2A somatic mutations identified to date (July 1996) in primary tumours. Underlined mutations appear in both ceillines and primary tumours. Information not specified in the original sources is indicated by (?). Abbreviations used in the table include:

paner: pancreas oesoph: oesophagus adeno: adenocarcinoma SCC: squamous cell carcinoma ALL: acute Iymphoblastic leukaemia (T-ALL: T-Iymphocyte ALL) CLL: chronic Iymphoblastic leukaemia NSCLC: non-small ceillung cancer HNSCC: head and neck squamous cell carcinoma B-NHL: B-Iymphocyte non-Hodgkin's Iymphoma HCC: hepatocellular carcinoma Table 4.2: CDKN2A somatie mutations-primary tumours

• TUMOUR BASE EVENT/CODON REF

EXON 1

? -17 dei 24 bp 1-3 PROSTATE 45 ? Ins C 4 MELANOMA 107 15 dei 37 bp 5 PANCR ADENO • 45 23 dei GCATGGA 8 ALL 108 /insTCCCGG 27G>A Mel911e HILAR BILE DUCT 45 33 dei 35 bp 11 B-NHL 109 42C>G Asp14Glu GALL BLADDER 45 47 del4 bp 16 PANCR ADENO • OESOPH SCC, 106,110, 111 47T>C Leu16Pro HILAR BILE DUCT 45 52 dei 32 bp 18 PANCRADENO 112 57 dei C 19 NSCLC 113 58 G>T Ala20Ser GALL BLADDER 45 59C>A Ala20Glu NSCLC 103 68 dei G 23 NSCLC 113 68G>A Gly23Asp PANCR· 45 71 G>C Arg24Pro SARCOMA 114 74T>C Val25Ala PROSTATE 115 78G>C Glu26Asp GALL BLADDER 45 ? dei 21 bp 29 PANCRADENO 112 88G>C Ala30Pro OESOPH SCC 45 97G>T Glu33STOP HNSCC 45 99G>T Glu33Asp HILAR BILE DUCT 45 109 C>T Leu37Leu MELANOMA 116 124 A>G Asn42Asp DUCTAL PANCR 96 132 C>A Tyr44STOP NSCLC 45 143 C>T Pro48Leu HNSCC 45 146 T>G lIe49Ser HILAR BILE DUCT 45 148 C>T Gln50STOP OESOPH SCC 110 150 GG>CC Gln50 Hisi MELANOMA 117 Val51 Leu

inll-l G>T splice NSCLC SCC 118 inl 1-8 dei 27 bp splice CLL 119 in11-9 del61 bp splice BLADDER 45 In11-2 A>T splice BLADDER 45

EXON2 151 G>A Val51Ile DUCTAL PANCR 96 152 T>A Val51Asp PANCRADENO 45 155 T>A Mel52Lys BREAST 45 157 dei A 53 BLADDER 45 158 T>C Mel53Thr CLL 119 160 dei A 54 PANCRADENO 112 164 dei G 55 OESOPHSCC 45 165 Ins 1 bp 55 ENDOMET 45 166 dei AG 56 NSCLCSCC 45 169 dei 13 57 B-NHL 109 171 dei CC 57 DUCTAL PANCR 96 • 170 C>T Ala57Vai ALL 45 172C>T Arg56STOP OESOPH SCC, BLADDER, 112,116 NSCLC SCC, PANCR ADENO 174 ins 7 bp 56 T-ALL 96 176 Ins G 59 NSCLCADENO 45 161 G>T Glu61 STOP OESOPH SCC, HNSCC 45 192del GCT 64 NSCLCSCC 45 194 dei 50 bp 65 OESOPHSCC 45 196 dei 1 bp His66STOP MELANOMA 107 (CAC>TA) 196 C>T His66Tyr NSCLC SCC 45 202G>A Ala66Thr OESOPH SCC 45 205 G>T Glu69STOP NSCLC 95 205 G>A Glu69Lys BLADDER 46 213 71 T-ALL 96 AAC>AAGGTCG 214 T>G Cys72Gly OESOPH SCC 45 216 delC 72 NSCLC LARGE CELL 116 216 C>A Cys72STOP NSCLC 95 217 G>A Ala73Thr GLiOBLASTOMA 120 220G>A Asp74Asn OESOPH SCC 45 221 A>T Asp74Vai HEPATIC BILE DUCT 45 224 C>T Pro75Leu BREAST 47 226G>A Ala76Thr OESOPH SCC 45 227 C>T Ala76Vai GLiOBLASTOMA 120 233" dei TC 1!! T-ALL 121 • 236 C>T Arg60STOP OESOPH SCC, NSCLC SCC, 45,99,11 BLADDER, T-ALL, PANCR·, ORAL 2,121 SCC 239 dei G 60 OESOPH SCC, B-NHL 45,109 239 G>T Arg60Leu HNSCC 45 242 C>T Pro61 Leu MELANOMA, THYROID 45,122 243 Ins 19 bp 62 PANCRADENO 112 247 C>A His63Asn NSCLC SCC 45 247 C>T His63Tyr HNSCC,PANCR·,BREAST 45 250 G>A Asp64Asn OESOPH SCC, NSCLC ADENO, 45 HNSCC 250 G>C Asp64His NSCLCADENO 45 250 G>T Asp64Tyr NSCLC SCC 45 252 C>A Asp64Glu BLADDER 46 253 G>A Ala65Thr GLiOBLASTOMA 120 257 ins G 66 NSCLC LARGE CELL 45 262 G>T Glu66STOP NSCLC SCC, MELANOMA 117,116 264 G>T Glu66Asp GALL BLADDER 45 266 dei GC 69 HCC 123 271" dei C 91 PANCR ADENO·, BLADDER 45,124 274 dei G 92 NSCLC SCC 45

• Because ofthe ambiguit)' in\'ol\'ed in assigning nucleotide positions 10 certain deletlons, these deletions are numbered from where the wild-type sequence lirsl changes. 277 A>G Thr93Ala NSCLCADENO 45 278 C>G Thr93Arg GLiOMA 45 284 T>C Val95Ala NSCLCADENO 45 292 C>T His98Tyr GLiOBLASTOMA 120 293 AC>CT His98Pro MELANOMA 45 293A>G His98Arg CLL 119 294 C>A His98Leu MELANOMA 45 296 G>A Arg99Gln NSCLCADENO 45 298 GC>CT Ala100Leu MELANOMA 45 305 C>T Ala102Vai GLiOBLASTOMA 120 307 dei CG/ins A 103 NSCLC SCC 118 310 dei C 104 OESOPHSCC 45 313 dei G 105 OESOPHSCC 45 314 dei 20 bp 105 PANCRADENO 45 316 G>A Val106Mel GLiOBLASTOMA 120 319 C>T Arg107Cys GLiOBLASTOMA 120 322 G>T Asp108Tyr HNSCC, NSCLC SCC 118 329 G>A Trp110STOP MELANOMA 45 330 G>A Trp110STOP MELANOMA, PANCR ., GLiOMA, 45,125 T·ALL 332 G>A Gly111Asp BLADDER 124 334 C>G Arg112 Gly MELANOMA 107 340 C>T Pro114Ser OESOPH SCC 45 341 C>T Pro114Leu ASTROCYTOMA 45 347 A>T Asp116Vai PROSTATE, CLL 115,119 350 dei T 117 BLADDER 124 352·440 88 bp dei 118 GLiOMA 120 355 G>C Glu119Gln GALL BLADDER 45 358 G>T Glu120STOP OESOPH ADENO, NSCLC 45 358 G>A Glu120Lys NSCLC ADENO, SCC 45 359 A>C Glu120Ala NSCLCADENO 45 364 G>A Gly122Ser AMPULLARY 45 365 dei G 122 NSCLC SCC 45 369 T>A His123Gln CLL 45 371 G>A Arg124His OESOPH SCC 45 374 dei A 125 T-ALL 121 375 T>C Asp125Asp BILE DUCT 45 378 C>T Val126Vai BLADDER 105 379 G>T Ala127Ser BLADDER 126 380 C>T Ala127Vai GLiOBLASTOMA 120 382 C>T Arg128Trp GLiOBLASTOMA 120 385 dei 23 bp 129 PANCRADENO 112 394 G>C Ala132Pro NSCLCADENO 45 401 C>T Ala134Vai NSCLCADENO 45 405 G>A Gly135Gly GALL BLADDER, GLiOMA, 95,98,12 NSCLC, STOMACH ,T·ALL 7,128 406 dei GG 136 OESOPH SCC 45 407G>A Gly136Asp GLiOBLASTOMA 120 424 C>T His142Tyr NSCLCADENO 45 430 dei C 144 OESOPHSCC 45 430 C>T Arg144Cys OESOPHSCC 45 449 G>T Gly150Vai NSCLC SCC 45 451 C>T Pro151Ser MELANOMA 116

xenograft SUMMARY • ûver the past two years, CDKN2A has been the subject ofintensive research. Clearly, CDKN2A is involvd in both sporadic and hereditary forms ofmelanoma. It is

also implicated in the development ofa wide variety oftumour types. This study has

investigated the role CDKN2A in hereditary predisposition to cancer by analysing

seventy-five families with excessive multiple cancers for germline CDKN2A mutations.

A previously reported Met531le alteration was detected in a family with numerous

cancers, including one case ofmelanoma. This result confirms that mutations in the

CDKN2A gene are not restricted to the hereditary melanoma syndrome FAMMM, and

thus CDKN2A mutations may predispose to many types ofcancers. However, CDKN2A

mutations are very rare or absent in families with multiple cancers that do not includc

melanoma. Therefore, future analyses for CDKN2A mutations should concentrate on

investigating families with: 1) individuals with multiple primaries, one of which includcs

melanoma, along with other cancers elsewhere in the fanüly and/or 2) cases with multiple

primaries and melanoma elsewhere in the family. Identification ofother families

harbouring CDKN2A mutations will provide further insights into the role ofthe cell cycle

inhibitor pléDKN2A in hereditary cancer and will hopefully lead 10 improved evaluation

ofcancer risks and more specific methods ofprevention and treatment ofmalignanl

disease.

47 CLAIMS TO ORIGINALITY • This thesis reports the identification ofa putative CDKN2A mutation in a family ascertained through the Adult Hereditary Cancer Clinic in Montreal. The work dcscribed

in this thesis has been completed by myself, unless otherwise stated.

48 PUBLICATIONS • ABSTRACTS Bignell, G., fuw.....S.., Biggs. P., Hamoudi. R., Rosenblatt, 1., Buu, P., Druker, H., Hudson. T., Houlston, R., Narod, S., Stratton, M., Foulkes, W.D. Non-medullary thyroid cancer: attempts to locate susceptibility genes by linkage in two Canadian families with goitre and non-medullary thyroid cancer. American Society ofHuman Genetics, 1996.

ARTICLES

Milner, B.1., Hosking, L., fuw.....S.., Haites, N.E., Foulkes, W.D. Polymorphisms in p21CIPI/WAFI arc not correlated with TP53 status in sporadic ovarian tumours. (European Journal ofCancer, in press).

~., Narod, S.A.. Aprikian, A., Ghadirian, P., Labrie. F. (1995) Androgen receptor and familial prostate cancer (Letter). Nalllre Medicine 1(9):848-849.

PRESENTATIONS

~., Narod, S.A., Foulkes, W.D. A pl6 mutation in a family with multiple cancers. Annual Meeting ofthe European Society ofHuman Genetics, April 11-13.1996, London. England.

~., Narod, S.A. Hereditary Multinodular Goitre. Hereditary Cancer Conference. University ofVermont, September 19, 1995, Burlington. Vermont.

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60 •

APPENDIX A: Pedigrees

The following abbreviations were used:

abdom: abdominal cancer bec: basal cell carcinoma bi: bilateral br: breast ca: cancer csu: cancer site unknown co: colon d: age at death dx: age at diagnosis GaSI: gall bladder Hodg: Hodgkin's disease ki: kidney lu: Jung Iym: Iymphatic Mmel: malignant melanoma NHL: non-Hodgkin's Iymphoma pan: pancreatic ca pro: prostate psu: primary site unknown sec: squamous cell carcinoma ste: stomach

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