Molecular genetics of colon cancer

Principal Investigator:

Margareta Nordling

Picture from: http://hubpages.com/hub/DNA-Testing-

for-Disease-Prevention

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Aims of the project

1. To make significant international contributions in elucidating the genetic

background of attenuated colon polyposis (AFAP), based on regional and national

patient cohorts.

2. To make personalized medicine possible for our patients, giving them a reliable

diagnosis, possibility of prognosis, adequate follow up, assessing the risk for

future generations and targeted therapy.

3. Contribute to increased understanding of variations in the genome and their effect

on the patient emphasizing the hereditary as well as the sporadic aspects of the

diseases, including investigations to find new disease-causing genes.

4. To implement advanced molecular genetics methodology in the clinic enabling

high-quality care for the patients.

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Background About 3 to 5% of people who develop colorectal cancer have an inherited genetic

susceptibility to the disease. Two syndromes are associated with the major part of

inherited colorectal cancer syndrome, hereditary non-polyposis colorectal cancer

(HNPCC) and familial adenomatous polyposis (FAP). In only 15-20% of the families

with HNPCC a deleterious mutation in one of the 3 predominant HNPCC genes can be

found. In families with FAP it is today possible to find almost all the mutations

responsible for the classical polyposis syndrome. However, in the attenuated form of the

disease (AFAP), with e.g. less polyps and higher age at onset, only a fraction of the

disease-causing mutations can be identified. The low detection rate implicates that

probably several responsible genes are still to be identified which also means that it is

important to study families from defined geographical regions as it might be that the

genetic cause of this disease is dependent on the origin of the families.

Elucidating the genetic background of hereditary cancer also has implications for the

understanding of the development of sporadic cancer. Besides the fact that germline

inactivation of APC is attributable to FAP, the gene is involved in the development of

sporadic colorectal cancers as it is inactivated in almost all sporadic colon tumours.

The gene has attracted vast interest since it encodes a multifunctional protein that

constantly is assigned new functions crucial for the function of the cell. Due to the

variable nature of the FAP syndrome extensive studies have been performed in trying to

elucidate the correlation between genotype and phenotype in this disease. The

manifestation of the disease can vary from the milder, attenuated form of disease (AFAP)

to a full classical phenotype with thousands of polyps with various degrees of

extraintestinal manifestations.

We and others have performed extensive studies of detecting mutations and performing

associations with specific genotypes and phenotypes in the FAP syndrome [1-4]. The

phenotypic expression of FAP is clearly dependant on the activity of the APC gene and

probably also on modifier genes. It has recently been shown that APC could be regulated

by alternative splicing perhaps thorough a mechanism involving cis-acting regulatory

RNA elements, trans-acting regulatory proteins and the NMD (non-sense mediated

decay) machinery. Although the functions of most spliced APC isofoms are not yet well

defined they could exert an antagonistic function versus the tumor-supressor activity of

the APC protein, thereby controlling tumorgenesis, differentiation and the development

of FAP [5].

FAP was originally assigned 100% penetrance in mutation carriers. However, the fact

that a very limited number of full mutation carriers, never develop any sign or are only

very mildly affected by the disease, has recently attracted attention. The possibility that

the FAP phenotype can be modulated by modifier genes has been proposed. To

understand how, and to what extend, the various mutations effect the function of the APC

mediated cell mechanisms, is important not only for studies on FAP, but also for the

general understanding of colon tumor development. For the same reason it is also crucial

to try to begin to understand how modifier genes can affect the disease phenotype. Today

we have the technical prerequisites to be able to perform experiments that could clarify

the differences in the genome, transcriptome and other factors affecting the expression

and function of the deleterious gene between mutation carriers that are affected by the

disease and those who are not.

The field of Clinical Genetics stands in front of a new era enabled by the introduction of

new sequencing technology. The recent introduction of Next-generation sequencing

(NGS) capable of producing millions of parallel DNA sequences is rapidly changing the

landscape of genetics, bringing new knowledge from basic biology to the genetic aspects

of personalised medicine. Already today and during the next few years there will be an

enormous focus on revealing the genetics behind hereditary disease, both rare and

common, and our unique patient material gives us a special opportunity to make

significant contributions to this research field.

Since instruments for NGS was introduced about three years ago, an increasing amount

of research publications have brought evidence for the usefulness of the technique also in

the direct practice of clinical genetics. The main sector of application for NGS in clinical

genetics (presently) is to find new disease-causing genes and to perform simultaneous

high-throughput analyses of an array of genes causing the same disease. Many more

applications such as detection of low-frequency alleles by ultra-deep NGS (meaning NGS

producing especially many sequence reads per sample) and detection of copy-number

variations are soon to come, improving the possibilities of personalized medicine both in

the case of hereditary cancer but also making analyses of somatic genetic variations in

sporadic cancer possible. These new possibilities will gradually replace the methodology

used in clinical genetics today and will also provide completely new knowledge about the

extent and significance of genetic variations in human diseases (both hereditary and

sporadic).

Material and methods Material

Patients and ethical approval

The majority of the patients included in the projects resides from the Cancer Genetic

Counselling Clinic at department of Oncology, SU/Sahlgrenska, department of Surgery,

Skaraborg Hospital and from the national Swedish Polyposis Registry, department. of

Medicine, Karolinska University Hospital.

The colon cancer project has very recently (august 2010) gained extended ethical

approval for the research connected to new sequencing and array methodology as this

methods holds the capacity to search the whole genome.

The research group and collaborations

The research group includes; one PhD student (AR) and one “biolog” both currently

working part time. We would like to extend the possibilities of having more lab personnel

and post-docs working in the project. The bioinformatics/biostatistic data generated in the

project is managed by Staffan Nilsson. Characterization of proteins will be performed by

Göran Karlsson. Targeted enrichment (e.g. exome enrichments) and NGS will initially be

outsourced to commercial labs e.g. GATC, Konstanz, Germany and others. The group of

From 2011 we will have the possibilities to perform these experiments in house (see

below).

Regional collaborations:

1. Dept of Surgery, Skaraborg Hospital (Stefan Skullman).

2. Doctors at several different hospitals in the Västra Götaland region based on

where the families live.

National collaborations;

1. The Swedish Polyposis Registry at the Dept. of Gastroenterology and Hepatology,

Karolinska University Hospital (Jan Björk).

International collaborations:

1. Barbara Graves, Dept of Oncological Sciences, University of Utah and Huntsman

Cancer Institute.

Equipment and research environment

The department of Clinical Genetics where the main part of the research is performed is

equipped with all basic and expensive instruments needed to conduct the basic research

of the project such as a variety of different PCR instruments, two ABI 3130xl sequencers

and an Affymetrix microarray scanner. Our sequencing capacity is already high but with

the availability of the facilities at Genomics Core Facility at SA, located in the same

building, we have extensive possibilities. The environment at the department of Clinical

Genetics provides extended knowledge of a variety of basic and sophisticated methods

e.g. mutation detection where a number of techniques are in daily use and various

microarray analyses etc. The Genomics Core Facility (located in the same building) also

provides access to a number of other instruments such as TaqMan for Q-PCR on the ABI

7900HT platform. In the beginning of 2011 (or earlier) an NGS platform including

instrumentation, laborative and bioinformatics support will be launched at the Genomics

Core Facility. The platform is a joint effort between the Sahlgrenska Academy and SU.

The main applicant (MN) of this research application is a member of the advisory group

for the establishment of the platform. Our department will be one of the main users of

this platform.

Methods

Mutation screening

For the next years to come the focus on development will be on NGS and its applications

but already today we have a number of different techniques for genome analyses running

in the lab that also will be used in the project. We perform mutation screening with a

variety of screening methods including e.g. MLPA (multiplex ligation-dependent probe

amplification) to detect deletions/duplications in genes and a variety of specific PCR-

based methods used to achieve as high mutation-detection frequency as possible (both on

DNA and cDNA level). Screening for mosaic mutations (using ultra-deep NGS) and

determination of lowered mRNA expression (microarray and Q-PCR) levels indicative of

mutations or epimutations are also performed. DNA sequencing (DNA and mRNA-

based) is used as a screening method but also for confirmation. We are also using MLPA

and bisulphate-modified sequencing to analyze for epigenetic modifications such as

aberrations in methylation pattern and we will also be analyzing for aberrations caused by

impaired histone acetylation. Using microarray (Affymetrix) analyses we presently detect

pathogenic CNVs and SNP in the genome. The SNP data is used for linkage analyses by

conventional methods and haplotype based homozygosity mapping. We are also using

this platform to analyze for specific transcript expression as described in Preliminary

results.

Next-generation sequencing

The NGS technique will bring us a number of new possibilities to perform genome

analyses. The NGS technique allows for whole-genome analyses and this was also the

purpose for developing the methodology. However, whole genome analysis is today not

feasible for most purposes due to cost, massive data handling and ethical considerations.

The technique is preferably used for analyzing targeted regions of the genome. Targeted

whole-genome exome sequencing where all coding exons in the genome are amplified

will be introduced as soon as possible. This method now begins to be widely used for

finding disease causing genes both in common and rare diseases. The methodology to

find new genes in this way includes massive bioinformatic handling, a service planned to

be provided by Genomics Core Facility but we also hope for possibilities to manage this

within the project. Staffan Nilsson from the dept of Statistics at Chalmers who is a

member of the research group will be important for this part of the project. Apart from

NGS a number of methods already running in the lab will be used when it comes to

characterize and select detected genetic variants.

We are already introducing ultra-deep NGS for detection of mutations that are only

present in a fraction of the cells of the patients, i.e. mosaic detection or low-frequency

allele detection. Before NGS there was no reliable method for this purpose. Our previous

study by Rohlin et al. [8] revealed detectable mutation levels down to 1 % using this

technique.

A number NGS applications will be used in the project ahead e.g. whole transcriptome

analyses and whole genome sequencing. A number of new databases built up from NGS

data, e.g. The 1000 Genomes Project

(http://en.wikipedia.org/wiki/1000_Genomes_Project, an international effort to establish

an overview of all genetic variations in humans compiled from 1000 human genomes)

and the Bejing Genome Institute (http://www.genomics.cn/en/index.php) make their data

freely available and we will also use these in the project.

Outline of the project. As suggested we will concentrate our future studies on a limited part of the project

as outlined below:

1. Identification of new genes causing AFAP

2. Studies on the genetic background of the variable expressivity of the diseases and

making an attempt to understand why some individuals are not affected by disease even

though they are full mutation carriers.

3. Contribution of inactivated tumor suppressor genes (e.g. the APC gene) to the

development of sporadic colon cancer. Does presence of low-frequency (mosaicism)

mutations in individuals contribute to the development of sporadic colon cancer?

Workplan 1. Identification of new genes

As described earlier we are interested in finding the genetic cause of disease for AFAP.

To accomplish this we have initiated analyses of patients using microarray-analyses both

for gene dosage (SNP 6.0 Array) and for transcript mapping (Exon 1.0 Array). Targeted

whole-genome exome sequencing with the NGS will be used to indentify causative genes

as outlined above. Apart from NGS a number of methods already running in the lab will

be used when it comes to characterize and select detected genetic variants. If linkage data

has to be improved we will use the high density arrays (Affymetrix 6.0 arrays) that are

now available. From in 2006 being able to genotype 10 000 SNPs on these arrays we

have today the possibility to genotype more than 900 0000 SNPs on one single array

which gives us much better possibilities to find the chromosomal location for a new

causative gene. We also have access to tumors which can be used to analyze for copy

number variants (CNV). Detection of CNVs compares intensities of CNV probes in

tumor DNA to probe intensities in normal DNA in a way to identify deleted or amplified

chromosomal regions in the genome. Deleted regions may indicate a tumor suppressor or

DNA repair gene involved in cancer development and amplified regions could be the

indication of an activated oncogene. We also use exon specific probes (as described in

section 3 below) to find varied expression patterns that could be due to an inactivated

disease-causing gene. Our intention is to investigate if patients without detectable

mutations possibly can carry new pathogenic isoforms from transcription of known

cancer-predisposing genes and also from other genes that could be implicated in tumor

development. In this way we will try to find clues to new mutations and predisposing

cancer genes. These studies are still on-going and will be continued during 2011.

2. Transcriptome studies using the Affymetrix GeneChip® Human Exon 1.0 ST

array in patients with variable expressivity of the diseases

The Human Exon 1.0 ST arrays contain approximately four probes per exon and roughly

40 probes per each known gene in the genome (over 5 million probes grouped into over

300,000 transcript clusters) and enables two complementary levels of analysis, total gene

expression from all known genes and alternative splicing from each specific gene. With

this platform it is possible to distinguish between different isoforms.

We will investigate if there are differences in expression from the whole genome in

families where a defined variable expression of disease is observed between family

members. In this way we will try to find out which genes that are able of modulating the

disease to be more severe or to be “silenced”. We will try to pin-point different genes or

isoforms that has an affect on the development of the disease in an attempt to try to

understand which mechanisms that triggers disease on-set and which that suppress

disease development. We have made the first analyses and are continuously collecting

samples for the study

3. APC mosaicism

We are using ultra-deep NGS to detect mosaic mutations in tumour, blood and normal

cell mucosa from patients with sporadic colon cancer. Our previous study by Rohlin et al.

[8] revealed detectable mutation levels down to 1 % using this technique. An additional

subject to study with this in background is the presence of epimutations that are

frequently mosaic within the individual, and inheritance in these cases is often weak.

Previous work and preliminary results Our previous work in the project is described in references [1, 3, 4, 6, 7, 8, 9].

1a. Identification of a novel mutation in Family 1 of the Swedish Polypos isRegistry.

In collaboration with The Swedish Polyposis Registry we have for several years been

working with the largest FAP family in Sweden in trying to find the disease-causing

mutation. This mutation has confounded us for many years and exhausted our

competence regarding mutation detection. Thanks to the high-density CNV arrays

(Affymetrix 6.0) that now are available we finally found the elusive disease-causing

mutation in spring this year. Initially we found one deletion far up-stream of the gene in a

region of no known regulatory significance for the APC gene. After a whole lot of

pondering and work we realized that the deletion was connected to a second deletion

separated by only 1500 base pairs impossible to detect on the array data. This second

deletion included approximately half of promoter 1B of the APC gene (Fig. 1). Apart

from being the first disease-causing mutation ever to be found in this region of the gene

the finding led to several new insights about the regulation of the APC gene that is

presented in the attached manuscript by Rohlin et al. [9]. Briefly, we had earlier observed

lowered expression of the disease causing allele in affected patients which led us to the

conclusion that promoter 1B is more important for the APC gene then generally

considered (which also was verified with expression studies). We could also suggest that

this lowered expression could protect carriers from development of desmoid tumours

which is a major cause of morbidity and mortality in FAP patients. Studies of adenomas

from the family also revealed interesting observations regarding the function of

inactivation of APC in tumorigenesis. As our project focuses on direct clinically relevant

issues the future studies regarding the function of APC in this family will be carried out

in collaboration with Prof Barbara Foulkes at Huntsman Cancer Institute, Utah. The APC

gene was identified at this institution many years ago and as the gene today is the most

studied gene in colon tumors both hereditary and sporadic, the Utah group has shown

considerable interest in our work.

Figure 1. Schematic illustration of the deletion in Family 1. The second deletion (Del 2)

includes 320 bp of promoter 1B (586 bp as a whole) of the APC gene.

1b. Identification of new genes

As described earlier we are interested in finding the genetic cause of disease for AFAP.

To accomplish this we have initiated analyses of patients using microarray-analyses both

for gene dosage (SNP 6.0 Array) and for transcript mapping (Exon 1.0 Array). Using

these methods we identified two genes (Fig. 2) with potential to be involved in the

disease. We will continue our studies of a large family from the Västra Götaland region

where we have localised a possible deleterious gene on chromosome 5 with a LOD score

of 2.7 and we will use the NGS approach including targeted amplification of exons with

subsequent bioinformatic filtering against known sequence variants to try to find the

causative gene. We are also continuously collecting families with this disease to use for

new-gene. These studies are still on-going and will be continued during 2011.

2. Patients with variable expressivity of the FAP diseases

We have made initial analyses using Affymetrix GeneChip® Human Exon 1.0 ST Array

on family members with reduced expression of the disease compared to members with a

classical polyposis. The analyses of these experiments are on going.

3. APC mosaicism As we established that very low levels of mosaic mutations could be detected [8] we

undertook a study on a material constituting of blood, tumour and normal tissue from

patients with sporadic colon cancer. The reason was that we previously had identified a

mosaic mutation in a patient with a very mild FAP disease [6], resembling sporadic colon

cancer, so our interest was to examine if sporadic colon cancer could be caused by

constitutional mosaic mutations. In fact, ultra-deep NGS did find very low levels of

mutation in blood and normal tissue in approximately 10% of the patients. The analyses

where performed by the Metagenomic facility at KTH, Stockholm. As these results are

very interesting and certainly will be questioned we decided to reanalyze some of the

samples at a different facility. These studies are now ongoing.

Figure 2. Partek GS alternative splice analyses of APC exon expression in AFAP

patients obtained from analyses with Affymetrix GeneChip Human Exon 1.0 ST Arrays.

Each blue dot represents the mean expression of an exon in the normal control (mean

value of three controls) and each red dot represents the mean expression of an exon in the

patient sample (mean value of three patients). The genes BTNL3 (A) and SPPL2B (B).

Significance and relevance of the project Knowledge gained from the studies of the hereditary variants of the diseases will

contribute to the understanding of initiation and development of sporadic tumors and in

this way we will try to acquire increased understanding of the underlying mechanisms for

initiation and development of colon tumors. The revolutionizing developments in genome

analyses during the last few years will bring us completely new tools to study our patient

material revealing new insights in diasease-related genetics bringing new knowledge in

how genetic variation is expressed in humans. Finding new cancer-predisposing genes

will be of importance, not only to families, but also for improved understanding of the

genetic factors underlying tumor development in general. By investigating the effect each

mutation have on the genes, the protein products and the function of the cell we hope to

contribute to a better understanding of the cancer related functions of the genes and in the

end we hope that this knowledge will lead to improved diagnostics and treatment for the

patients.

References: 1. Bjork J, Akerbrant H, Iselius L, Bergman A, Engwall Y, Wahlstrom J, Martinsson T,

Nordling M, Hultcrantz R: Periampullary adenomas and adenocarcinomas in familial

adenomatous polyposis: cumulative risks and APC gene mutations. Gastroenterology 2001, 121(5):1127-1135.

2. Jass JR: Colorectal polyposes: from phenotype to diagnosis. Pathology, research and

practice 2008, 204(7):431-447. 3. Kanter-Smoler G, Bjork J, Fritzell K, Engwall Y, Hallberg B, Karlsson G, Gronberg H,

Karlsson P, Wallgren A, Wahlstrom J, Hultcrantz R, Nordling M: Novel findings in

Swedish patients with MYH-associated polyposis: mutation detection and clinical

characterization. Clin Gastroenterol Hepatol 2006, 4(4):499-506. 4. Nordling M, Engwall Y, Wahlstrom J, Wiklund L, Eriksson MA, Gustavsson B, Fasth S,

Larsson PA, Martinsson T: Novel mutations in the APC gene and clinical features in

Swedish patients with polyposis coli. Anticancer Res 1997, 17(6D):4275-4280.

5. De Rosa M, Scarano MI, Panariello L, Carlomagno N, Rossi GB, Tempesta A, Borgheresi P, Renda A, Izzo P: Three submicroscopic deletions at the APC locus and

their rapid detection by quantitative-PCR analysis. Eur J Hum Genet 1999, 7(6):695-703.

6. Kanter-Smoler G, Fritzell K, Rohlin A, Engwall Y, Hallberg B, Bergman A, Meuller J, Gronberg H, Karlsson P, Bjork J, Nordling M: Clinical characterization and the

mutation spectrum in Swedish adenomatous polyposis families. BMC medicine 2008,

6:10. 7. Meuller J, Kanter-Smoler G, Nygren AO, Errami A, Gronberg H, Holmberg E, Bjork J,

Wahlstrom J, Nordling M: Identification of genomic deletions of the APC gene in

familial adenomatous polyposis by two independent quantitative techniques. Genet Test 2004, 8(3):248-256.

8. Rohlin A, Wernersson J, Engwall Y, Wiklund L, Bjork J, Nordling M: Parallel

sequencing used in detection of mosaic mutations: comparison with four diagnostic

DNA screening techniques. Hum Mutat 2009, 30(6):1012-1020. 9. Rohlin et al., Alteration of promoter 1B of APC in classical adenomatous polyposis-

predictor of low desmoid risk. Submitted