Post-zygotic Genetic Variation in Health and Disease609581/FULLTEXT01.pdfPost-zygotic genetic variation, twins as a model in studies of post zygotic variation and the methods involved

ACTAUNIVERSITATIS

UPSALIENSISUPPSALA

2013

Digital Comprehensive Summaries of Uppsala Dissertationsfrom the Faculty of Medicine 873

Post-zygotic Genetic Variation inHealth and Disease

HAMID REZA RAZZAGHIAN

ISSN 1651-6206ISBN 978-91-554-8614-3urn:nbn:se:uu:diva-196217

Dissertation presented at Uppsala University to be publicly examined in Rudbecksalen,Rudbeck Laboratory, Dag Hammarskjölds väg 20, Uppsala, Tuesday, April 23, 2013 at09:15 for the degree of Doctor of Philosophy (Faculty of Medicine). The examination will beconducted in English.

AbstractRazzaghian, H. R. 2013. Post-zygotic Genetic Variation in Health and Disease. ActaUniversitatis Upsaliensis. Digital Comprehensive Summaries of Uppsala Dissertations fromthe Faculty of Medicine 873. 80 pp. Uppsala. ISBN 978-91-554-8614-3.

Post-zygotic genetic variation has previously been shown in healthy individuals and linkedto various disorders. The definition of post-zygotic or somatic variation is the existence ofgenetically distinct populations of cells in a subject derived from a single zygote. Structuralchanges in the human genome are a major type of inter-individual genetic variation and copynumber variation (CNV), involving changes in the copy number of genes, are one of the beststudied category of structural genetic changes. In paper I we reported a pair of healthy femalemonozygotic (MZ) twins discordant for aneuploidy of chromosomes X and Y, contributing to thedelineation of the frequency of somatic variation in MZ twins. It also illustrates the plasticity ofthe genome for tolerating large aberrations in healthy subjects. In paper II we showed age-relatedaccumulation of copy number variation in the nuclear genomes in vivo for both megabase- andkilobase-range variants. Using age-stratified MZ twins and single-born subjects, we detectedmegabase-range aberrations in 3.4% of people ≥60 years old but not in individuals youngerthan 55 years. Moreover, the longitudinal analysis of subjects with aberrations suggests that theaberrant cell clones are not immortalized and disappear from circulation. We also showed thatsorted blood cells display different genomic profiles. The detected recurrent rearrangementsare candidates for common age-related defects in blood cells. This work might help to describethe cause of an age-related decline in the number of cell clones in the blood, which is one of thehallmarks of immunosenescence. In paper III we described a variable number tandem repeat(VNTR) ~4 kb upstream of the IFNAR1 gene, which was somatically variable. We detected14 alleles displaying inter- and intra-individual variation. Further analyses indicated strongclustering of transcription factor binding sites within this region, suggesting an enhancer. Thisputative VNTR-based enhancer might influence the transcriptional regulation of neighboringcytokine receptor genes and the pathways they are involved in.

These three studies stress the importance of research on post-zygotic variation in genetics.Furthermore, they emphasize that biobanks should consider sampling of multiple tissues tobetter address this issue in the genetic studies.

Keywords: Post-zygotic genetic variation, monozygotic twins, copy number variation, singlenucleotide polymorphism, variable number tandem repeat

Hamid Reza Razzaghian, Uppsala University, Department of Immunology, Genetics andPathology, Medical Genetics, Rudbecklaboratoriet, SE-751 85 Uppsala, Sweden.

© Hamid Reza Razzaghian 2013

ISSN 1651-6206ISBN 978-91-554-8614-3urn:nbn:se:uu:diva-196217 (http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-196217)

The important thing is not to stop questioning Albert Einstein (1879-1955)

To my family

List of Papers

This thesis is based on the following papers, which are referred to in the text by their Roman numerals.

I Razzaghian HR*, Shahi MH*, Forsberg LA*, de Ståhl TD, Ab-sher D, Dahl N, Westerman MP, Dumanski JP. (2010) Somatic mosaicism for chromosome X and Y aneuploidies in monozygot-ic twins heterozygous for sickle cell disease mutation. Am J Med Genet A, 152A(10):2595-8.

II Forsberg LA, Rasi C, Razzaghian HR, Pakalapati G, Waite L, Thilbeault KS, Ronowicz A, Wineinger NE, Tiwari HK, Boomsma D, Westerman MP, Harris JR, Lyle R, Essand M, Eriksson F, Assimes TL, Iribarren C, Strachan E, O'Hanlon TP, Rider LG, Miller FW, Giedraitis V, Lannfelt L, Ingelsson M, Pi-otrowski A, Pedersen NL, Absher D, Dumanski JP. (2012) Age-related somatic structural changes in the nuclear genome of hu-man blood cells. Am J Hum Genet, 90(2):217-28.

III Razzaghian HR*, Forsberg LA*, Prakash KR, Przerada S, Pap-rocka H, Zywicka A, Westerman MP, Pedersen NL, O'Hanlon TP, Rider LG, Miller FW, Srutek E, Jankowski M, Zegarski W, Piotrowski A, Absher D, Dumanski JP. (2013) Post-zygotic and inter-individual structural genetic variation in a putative enhancer element of the locus between the IL10Rβ and IFNAR1 genes. Manuscript.

*Authors contributed equally to the work.

Reprints were made with permission from the respective publishers.

Other papers by the author:

IV Conze T, Göransson J, Razzaghian HR, Ericsson O, Oberg D, Akusjärvi G, Landegren U, Nilsson M. (2010) Single molecule analysis of combinatorial splicing. Nucleic Acids Res, 38(16):e163.

V Badhai J, Fröjmark AS, Razzaghian HR, Davey E, Schuster J, Dahl N. (2009) Posttranscriptional down-regulation of small ri-bosomal subunit proteins correlates with reduction of 18S rRNA in RPS19 deficiency. FEBS Lett, 583(12):2049-53.

Contents

Introduction ................................................................................................... 13 Genetic variation ...................................................................................... 13

Single nucleotide polymorphisms (SNPs) ........................................... 14 Structural genetic variation and copy number variation (CNV) .......... 14 Tandem repeats (TRs) .......................................................................... 17 Epigenetics ........................................................................................... 18

Post-zygotic mosaicism ............................................................................ 19 Post-zygotic genetic variation in health and disease ................................ 23 Twins in genetic research ......................................................................... 25 Genetics of aging ...................................................................................... 29 Blood cells, immune system and genotyping ........................................... 30 Encyclopedia of DNA Elements (ENCODE) .......................................... 32 Interferons and interferon receptors ......................................................... 33

Methods ........................................................................................................ 35 Array comparative genomic hybridization (aCGH) ................................. 35 Illumina single nucleotide polymorphism genotyping ............................. 36 Quantification of percentage of aberrant cells carrying structural variation .................................................................................................... 39

Fluorescence in situ hybridization (FISH) ........................................... 39 Quantitative real-time polymerase chain reaction (qPCR) .................. 39 Mosaic alteration detection (MAD) ..................................................... 40

Present investigations .................................................................................... 41 Aims ......................................................................................................... 41 Paper I. Somatic mosaicism for chromosome X and Y aneuploidies in monozygotic twins heterozygous for sickle cell disease mutation ........... 42

Introduction ......................................................................................... 42 Results and discussion ......................................................................... 42

Paper II. Age-related somatic structural changes in the nuclear genome of human blood cells ................................................................................ 43


Paper III. Post-zygotic and inter-individual structural genetic variation in a putative enhancer element of the locus between the IL10Rβ and IFNAR1 genes .......................................................................................... 48


Concluding remarks and future perspectives ................................................ 51

Acknowledgements ....................................................................................... 53

References ..................................................................................................... 55

Abbreviations

aCGH Array comparative genomic hybridization ADHD Attention-deficit hyperactivity disorder AIDS Acquired immunodeficiency syndrome AK2 Adenylate kinase 2 ASPE Allele-specific primer extension aUPD Acquired uniparental disomy BAF B-allele frequency Bdev B deviation bp Base pair CBS Circular binary segmentation CD Cluster of differentiation cDNA Complementary DNA ChIP-seq Chromatin immunoprecipitation sequencing CNNLOH Copy number neutral loss of heterozygosity CNV Copy number variation CR Caloric restriction Ct Cycle threshold CTL Cytotoxic T lymphocyte DM1 dystrophia myotonica 1 DM1PK dystrophia myotonica 1 protein kinase DMD Duchenne muscular dystrophy DNA Deoxyribonucleic acid DSB double-strand break DZ Dizygotic FISH Fluorescence in situ hybridization FoSTeS Fork stalling and template switching FRAXA Fragile X syndrome gDNA Genomic DNA GWAS Genome-wide association study HCV Hepatitis C virus HD Huntington disease HPV Human papillomatosis virus IFN Interferon IFN-α Interferon alpha IFN-β Interferon beta IFN-γ Interferon gamma IFN-λ Interferon lambda

IFNAR Interferon alpha, beta and omega receptor IFNAR1 Interferon alpha, beta and omega receptor 1 IFNAR2 Interferon alpha, beta and omega receptor 2 IFNGR1 Interferon gamma receptor 1 IFNGR2 Interferon gamma receptor 2 IFN-λR1 Interferon lambda receptor 1 Ig Immunoglobulin IGF2 Insulin-like growth factor II IL10Rβ Interleukin 10 receptor beta iNOS Incudible nitric oxide INS Insulin kb Kilobase KS Kaposi’s sarcoma LCR Low copy repeat LD Linkage disequilibrium LOH Loss of heterozygosity LRR Log R ratio MAD Mosaic alteration detection Mb Megabase MDA Multiple displacement amplification MDS Myelodysplastic syndrome MHC Major histocompatibility complex MS Multiple sclerosis mtDNA Mitochondrial DNA MZ Monozygotic NAHR Non-allelic homologous recombination NF1 Neurofibromatosis type 1 NHEJ Non-homologous end joining NK cells Natural killer cells nt Nucleotide PCR Polymerase chain reaction qPCR Quantitative real-time PCR RA Rheumatoid arthritis RNA Ribonucleic acid SBE Single-base extension SBMA Spinobulbar muscular atrophy SCID Severe combined immunodeficiency SLE Systemic lupus erythematosus SNP Single nucleotide polymorphism SSR Simple sequence repeat STr Sickle cell trait STR Short tandem repeat TCR T cell receptor Th cells T helper cells TR Tandem repeat

UCE Ultraconserved element ULSAM Uppsala longitudinal study of adult men UPD Uniparental disomy VNTR Variable number tandem repeat WGA Whole genome amplification WGG Whole genome genotyping

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Introduction

The human genome consists of 20000-25000 protein coding genes [1] com-prising approximately 1.5% of the human genome. After completion of the human genome project, many efforts were focused on identification of nor-mal inter-individual genetic variation and discovery of correlation between a disease phenotype and a specific genotype. Different types of genetic varia-tion such as single nucleotide polymorphisms (SNPs) and structural changes in the human genome will be discussed in the first part of the thesis. High resolution analysis using various microarray-based methods such as array-based comparative genomic hybridization (aCGH) and SNP arrays has led to significant improvements of our understanding of various types of genetic variation. These genome-wide tools enable scientists to characterize genetic variants even when only a minority of cells is affected by a genetic differ-ence. Post-zygotic genetic variation, twins as a model in studies of post-zygotic variation and the methods involved are topics that will be covered in the second part of the thesis.

Genetic variation Genetic variation is diversity in DNA sequence in the genome of individuals in populations [2]. When frequency of a genetic variant is considered, the variants at a specific locus which are present in >1% of subjects in the popu-lation, are called polymorphisms. The rare variants, which occur with fre-quency of less than 1% are commonly classified as mutations [3]. Completion of the draft of human genome sequence in 2001 had a signifi-cant impact on expanding our understanding of human genetic variation [4, 5]. During the recent years, genetic research has focused on elucidation of correlation between human genetic variation and phenotypic diversity [3]. Initial studies concentrated mainly on the patterns and frequencies of single nucleotide polymorphisms (SNPs) among diverse populations [6-8]. After discovery of structural genetic variation [9, 10], the focus of studies has ex-panded also towards interpretation of this type of variants [11-14]. Howev-er, our knowledge about distribution and frequency of structural variants in the genome is still inadequate and cataloguing these variants is an important

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aspect towards better understanding of the level of normal variation and their implication on disease phenotypes [3]. Application of massively parallel sequencing technology has further led to identification of novel structural genetic variants in the human genome [15-17]. Variation in the human DNA can be divided into many categories and will be discussed in the following sections.

Single nucleotide polymorphisms (SNPs) For approximately a decade, it has been thought that SNPs are the most pre-dominant type of genetic variation between unrelated subjects [3, 18]. SNP is the difference in the DNA sequence in the position of a single nucleotide. Initial sequencing-based research estimated that the human genome contains at least 11 million SNPs, comprising nearly 0.3% of the genome [3]. In a recent, more extensive report within “The 1000 genomes project”, a validat-ed haplotype map of 38 million SNPs among 1092 human genomes was identified [19]. The impact of SNPs on phenotypes usually depends on if polymorphism occurs in the coding or non-coding positions in the DNA sequence [3, 20-22]. SNPs have been used for identification of disease-predisposing loci in the context of genome-wide association studies (GWASs). GWASs focus on identification of SNPs at different genetic loci in the genome that are associated with disease. GWAS rely on the principle of linkage disequilibrium (LD) at the population level and are based upon LD between genotyped SNPs and ungenotyped variant that may have a func-tional consequence on disease risk [23]. There exists a strong correlation between the proportions of discovered variants with the sample size in GWASs. During the recent years, GWAS had a significant impact in identi-fication of predisposing loci to numerous diseases, for instance rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE) [23]. However, SNP-based variation in the human genome is not the focus of this thesis and will not be further discussed.

Structural genetic variation and copy number variation (CNV) Structural changes in the human genome (i.e. deletions, gains/insertions, translocations, inversions and complex rearrangements) have been identified as a major type of inter-individual variation [14]. Furthermore, the rate of de novo formation for structural variants in the germ-line exceeds the corre-sponding rate for single nucleotide polymorphisms (SNPs) by a few orders of magnitude [24, 25]. The best explored subtype of structural genetic vari-

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ants involves changes affecting copy number of DNA segments or copy number variation (CNV). CNVs can affect chromosomal segments larger than 1 kilobase (kb) to many megabases (Mb), including entire chromo-somes (aneuploidy), which are variable in copy number when compared to the reference genome [26]. As mentioned previously, CNVs were discov-ered more recently compared to SNPs [9, 10] and due to the short period since their discovery, when compared to SNPs, still are often not considered as the main type of genetic variation in humans. Segmental duplications or low copy repeats (LCRs) are blocks of usually tandemly repeated genomic DNA sequence, typically between 10 to 300 kb in size, which are present in two or more genomic locations. They are de-fined as sequence blocks with more than 90% sequence identity with each other, but it is not unusual that the degree of sequence identity between blocks of sequences within segmental duplications is more than 95%, or even higher. This category of sequences encompasses ~5% of the sequence of the human genome [26-29] and they are important because of their in-volvement in the generation of CNVs, via the process of non-allelic homolo-gous recombination (see below). Copy number neutral loss of heterozygosity (CNNLOH, also called unip-arental disomy; UPD) represents another special type of structural genetic variation. The definition of CNNLOH for a single chromosome is the inher-itance of both homologues of a pair of chromosomes from a single parent [30]. CNNLOH does not alter the copy number of affected chromosomal segment. CNNLOH is, however, a result of a structural rearrangement, and most commonly arise due to meiotic or mitotic non-disjunction/anaphase lag, alternatively mitotic recombination [31]. CNNLOH can affect the entire chromosome or smaller, usually terminal chromosomal segments (segmental CNNLOH or segmental UPD). There are two major consequences of CNNLOH from the point of view of disease; an imprinting disorder via loss of imprinting or inheritance of a recessive trait in a non-Mendelian fashion. The second is due to “reduction to homozygosity” leading to a recessive phenotype to appear, which is inherited from a heterozygous parent. CNNLOH has been shown to be involved in the development of cancers and developmental disorders. The list of conditions that have been associated with CNNLOH is continuously growing [30, 32-34] and this trend is likely to continue due to application of SNP-based arrays with high resolution of analysis. Copy number variation includes deletions, duplications and insertions [26]. They can be inherited through the germline or arise post-zygotically as a change acquired during lifetime [11, 26, 35, 36]. Recent estimations suggest that up to 12% of the genome is subject to CNV [11, 35]. CNVs are not

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randomly distributed in the human genome, but tend to be clustered in re-gions of complex genomic architecture, which comprise complex patterns of segmental duplications [35]. It has been reported that MZ twins can have different CNVs profiles [37] and differentiated human tissues from the same individual can vary in copy number [38]. Changes in copy number might alter the expression levels of genes included in the regions of variable copy number [35]. Various CNVs were linked with complex traits, such as asso-ciation of a 45-kb deletion upstream of NEGR1 (neuronal growth regulator 1) with body mass index [14, 39], a 117-kb deletion of UGT2B17 (UDP glucuronosyltransferase 2 family, polypeptide B17) with osteoporosis [14, 40] and a 20-kb deletion upstream of IRGM (immunity-related GTPase fami-ly, M) with Crohn’s disease [14, 41].

Mechanisms of CNV formation Three major mechanisms for generation of genomic rearrangements in the human genome have been described: non-allelic homologous recombination (NAHR), non-homologous end joining (NHEJ) and fork stalling and tem-plate switching (FoSTes). Each of these is related to different normal genet-ic processes, such as recombination, DNA repair and replication, respective-ly.

Non-allelic homologous recombination (NAHR) The most recurrent genomic rearrangements are results of NAHR between two segmental duplications. Due to the high sequence similarity, non-allelic copies of segmental duplications can sometimes be misaligned in meiosis or mitosis. This misalignment and the following crossover between them can cause genomic rearrangements in progeny cells. When the two segmental duplications are situated on the same chromosome and in direct orientation, NAHR between them result in duplication and/or deletion. To the contrary, when segmental duplications are positioned on the same chromosome but in opposite orientation, NAHR leads to inversion of the fragment flanked by them. NAHR between repeats on different chromosomes can cause translo-cation [29].

Non-homologous end joining (NHEJ) NHEJ is a main mechanism for repair of double-strand breaks (DSBs) in eukaryotic cells. This process is also considered as the main mechanism responsible for rejoining of translocated chromosomes in cancer. NHEJ

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consists of four steps: detection of DSB, bridging of both DNA ends, modi-fication of the ends for ligation and ligation as the final step. NHEJ exhibits two specific features. The first is that segmental duplications are not re-quired for occurrence of NHEJ. Secondly, addition or deletion of nucleo-tides at the rejoining site can take place. It has been proposed that NHEJ breakpoints often located within repetitive elements [29].

Fork stalling and template switching (FoSTes) FoSTes takes place when the DNA replication fork stalls at one position followed by disengagement of the lagging strand from the original template. Next, the lagging strand transfers and then anneals, by virtue of microho-mology at the 3’ end, to another replication fork in physical vicinity. Then, the annealed strand primes its own template-driven extension at the trans-ferred fork. Switching to a new fork located downstream would lead to a deletion whereas if the new fork situated upstream the result will be a dupli-cation. Furthermore, depending on the direction of the fork progression and whether the lagging or leading strand in the new fork was used as template and copied, the erroneously incorporated fragment from the new fork could exhibit direct or inverted orientation compared to its origin. The entire pro-cess can arise multiple times in series leading to complex rearrangements [29].

All of the above briefly described mechanisms can arise in germ cells, where the rearrangements can be linked to genomic disorders as well as in somatic cells where the rearrangements can also result in disorders, for instance can-cer [29].

Tandem repeats (TRs) DNA repeats fall into two main categories: interspersed repeats and TRs. The interspersed repeats are remnants of transposons that are interspersed throughout the genome while in TRs, the repeat units are positioned next to each other [42]. TRs possess a highly polymorphic nature and are useful in many areas of biomedicine, for instance in forensic studies [43] and in clin-ics to identify recipients of bone marrow transplants [44]. Repeats encom-pass 46% of the human genome [42]. The de novo mutation rate for TRs is very variable; the numbers between 10-3 and 10-7 per locus per cell division have been suggested. This indicates that TRs are extremely variable and often display 10 to 100,000 times higher mutation rate compared to other parts of the genome [42]. The mutation frequency of TRs depends on the length and number of repeat units as well as purity of repeat tract. However,

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the most important factor defining a TR’s variability appears to be the num-ber of repeat units [42]. Due to their variability, TRs are also known as vari-able number tandem repeat (VNTR) elements. VNTRs are divided into two groups: 1) Microsatellites (also called simple sequence repeats; SSRs, or short tandem repeats, STRs), which are repeats with unit length of <10 nu-cleotides (nt); and 2) Minisatellites with units ≥10 nt in length [42].

Approximately 17% of protein coding genes in the human genome contain repeats within transcribed regions [42]. Various diseases such as Fragile X syndrome (FRAXA) [45], spinobulbar muscular atrophy (SBMA) [46] and Huntington disease (HD) are associated with the repeats. The common ge-netic feature in these three is the expansion of the number of TRs located within a specific gene and affected individuals with greater TR expansions display earlier onset and higher severity of the disease [42]. TRs also cause variation in gene expression. For instance, shorter alleles of TRs upstream of the human insulin (INS) gene are associated with alteration in expression of INS and predisposition to insulin-dependent diabetes mellitus [47]. Fur-thermore, the same TR influences on the expression of the nearby insulin-like growth factor II (IGF2) gene [48]. Another example is increase in the transcription of incudible nitric oxide (iNOS) with increasing number of CCTTT repeats in its promoter [49].

Epigenetics The term “epigenetics” denotes all (via meiosis as well as via mitosis) herit-able changes in gene function, which are not encoded in the DNA sequence itself [50]. Epigenetic changes allow alleles, cells or even individuals that are genetically identical to display different phenotypes [51]. Epigenetic variation could lead to silencing or activation of a specific gene in a specific tissue resulting in a phenotypic variation [52]. There are two main mecha-nisms of epigenetic modification in eukaryotic cells. First, is the methyla-tion of cytosine in CpG di-nucleotides occurring mainly at CpG islands, which are dense in CpG di-nucleotides [51]. Promoter-associated CpG is-land methylation serves as one level of epigenetic control, which cooperates with histone modifications (see below) to reach molecular activation or si-lencing [52-54]. For instance, if a single methylation site was not methylat-ed after DNA replication, the presence of methylated sites in the vicinity as well as modified histones will lead to remethylation of the site at a later time [51]. Methylation of CpG islands in the promoter regions has been intro-duced as a common defect in cancer cells [55]. Furthermore, population-specific methylation patterns of DNA have been reported [56]. The second major mechanism of epigenetic control of gene function includes different modifications of histones [51]. Histone modifications are associated with

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transcriptional state of neighboring genes. Acetylation of lysine in histones almost always associates with chromatin accessibility and transcriptional activity (euchromatin) while lysine methylation can have diverse effects depending on which residue is modified. For instance, methylation of H3K4 (methylation of the lysine at position four of the H3 histone) and H3 lysine 36 is correlates with transcribed chromatin. In contrary, methylation of H3 lysine 9 (H3K9) and H3 lysine 27 (H3K27) show a relationship with repres-sion and a more dense chromatin structure (heterochromatin) [57].

In summary, I discussed above different types of genetic and epigenetic var-iation. The recent literature points out that CNV-based variation is more common than variation due to base substitutions [24, 25]. It has also been described that copy number variable regions encompasses more nucleotide content per genome than SNPs, emphasizing the significance of CNV in genetic diversity [11]. This thesis focuses on the structural genetic variation including CNVs that arise post-zygotically and is acquired during lifetime.

Post-zygotic mosaicism

Analyses of various types of human genetic variation performed hitherto are heavily dominated by comparisons of different people. Little attention has been paid to analysis of acquired during life-time differences between nor-mal somatic cells from different organs/tissues of the same person (i.e. post-zygotic variation, or mosaicism), either in healthy subject or in people af-fected by a disease [58].

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Figure 1. Three major classes of post-zygotic large-scale structural genetic aberra-tions. Panels A, B and C show a deletion, a copy number neutral loss of heterozy-gosity (CNNLOH, also named acquired uniparental disomy, aUPD), and a gain, respectively. Each panel shows images from Illumina single nucleotide polymor-phism (SNP) arrays showing a selected abnormal chromosome, with the affected regions emphasized in pink. The results from Illumina SNP arrays consist of two data tracks: log R ratio (LRR) values of fluorescent intensities from array probes (upper part), and B allele frequency (BAF) values representing the fraction of fluo-rescent intensity at each SNP accounted for by the B allele (lower part). In normal situation, BAF values cluster around 0 (AA genotype), 0.5 (AB) or 1 (BB). On the right hand side, a schematic explanatory figure shows the mosaic mixture of cells with aberrant and wild-type chromosomes. Two hypothetical homologous chromo-somes (labelled in green and white) with heterozygous genotypes for six SNPs are shown. Panel A shows data for chromosome 5 in a monozygotic (MZ) twin pair sampled at the age of 77 years. MZ twin TP25-1 has a normal profile, while its co-twin TP25-2 exhibits a 32.5 Mb deletion on 5q in approximately 55% of nucleated blood cells. This deletion is discovered using both LRR (downward shift) and BAF (heterozygous SNPs cluster away from 0.5) data from the Illumina SNP chip. The Illumina profile consists of a mixture of genotypes from aberrant cells (roughly 55%) and wild-type cells representing approximately 45% of nucleated blood cells. Panel B displays data for chromosome 10 in MZ twin pair sampled at the age of 77 years. Twin TP12-1 shows a normal profile. Utilizing BAF values, a 76.5 Mb large CNNLOH/aUPD was identified on 10q in co-twin TP12-2. Quantification of cells carrying the CNNLOH/aUPD suggests that 34% of cells have aberrant genotype. As this aberration does not change the copy number of the aberrant segment, LRR val-ues are normal. However, the genotypes of SNPs within this segment are all homo-zygous in aberrant cells. Panel C illustrates data for chromosome 8 from subject ULSAM-298 using two samples collected at the ages of 71 and 88 years. The sam-ple collected at 71 years is normal, while the sample taken at the age of 88 years shows a 70 Mb gain of chromosome 8 in roughly 30% of cells, visible with both LRR and BAF data from the Illumina chip (Figure taken from Forsberg LA, Absher D, Dumanski JP. Non-heritable genetics of human disease: spotlight on post-zygotic genetic variation acquired during lifetime. Journal of medical genetics 2013;50(1):1-10, with permission).

The definition of post-zygotic or somatic variation is the existence of genet-ically distinct populations of somatic cells in an individual derived from a single zygote [59]. Mosaicism can arise in both clones of somatic and germ-line cells. Mosaicism in cellular populations can occur due to point muta-tions in nuclear or mitochondrial (mt) DNA in post-zygotic cells, epigenetic modifications of DNA, and numerical or structural aberrations of chromo-somes [59]. Somatic mosaicism has been established as a cause of miscar-riage, birth defects, developmental delay and cancer [60-66]. The paucity of studies on somatic post-zygotic mosaicism is remarkable, especially in view of the fact that the common human disorders are apparently not due to inher-itance of defective allele(s) from the parents and this is in agreement with the fact that only a limited proportion of heritable component of any complex

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trait has been identified [67]. Recent review from our group suggested that post-zygotic variation may explain a substantial part of the cause of non-heritable diseases [58]. Other reviews have also pointed out that somatic mosaicism is understudied and consequently underestimated [31, 58, 59, 68, 69]. Furthermore, theoretical predictions suggest that the somatic variation must be widespread [70-72]. Estimations suggest that an adult human body contains 1013-1014 cells and approximately 1016 cells are produced during human lifetime. There is possibility for occurrence of mutation in each so-matic cell division [70-72]. Michael Lynch in his recent review from 2010 [71] states:

…with a human germ-line mutation rate of ∼10−8 base substitu-tions/site/generation, a site in a somatic nucleus will be mutated with a prob-ability of 10−7 to 10−6 by the average age of reproduction, with the burden be-ing higher in older individuals. With a diploid genome size of 6×109 sites and ∼1013 cells per soma, the body of a middle-aged human might then con-tain >1016 mutations (not including insertions, deletions, or other larger scale mutations). Only about 1% of the human genome consists of coding DNA, so a substantial fraction of somatic mutations will be inconsequential, but even if just 1% of coding mutations had significant fitness effects, the total body burden of mutations would be of order 1012.

The above estimations have been calculated using studies of SNPs and not include structural variants. It should be considered that although structural variants are less well characterized compared to single nucleotide polymor-phisms, they are estimated to be more frequent compared to single nucleo-tide polymorphisms [58]. So, inclusion of results from studies on structural variation will likely dramatically increase the total body burden of muta-tions. Reviews suggest that the mutation frequency for the base substitution per cell division in somatic cells is 4-25 times greater than the corresponding frequency for the germline [58, 72] and the germline mutation rate of CNVs is more common than the corresponding rate for SNPs by a few orders of magnitude [24, 25]. Therefore, the predicted frequency of post-zygotic mu-tations in the human soma during a single lifetime is vast. Three main types of structural genetic changes; deletions, gains and copy number neutral loss of heterozygozity (CNNLOH, also known as acquired uniparental disomy, aUPD) are known to occur post-zygotically and are schematically shown in figure 1. Comparison of frequency for these three main types of mega-base range structural mutations indicates that deletions are more common than gains. Although it might be difficult to distinguish between CNNLOH/aUPD and a gain or deletion event in case where only about 10% of cells are affected, CNNLOH/aUPD are the most frequent or the second most frequent structural variation and represent between 22% and 48% of all mutations [58, 73-75].

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In paper II, we detected megabase-range copy-number differences in blood DNA of ~3% of the studied subjects using both MZ twins and single-born individuals from ULSAM cohort [73]. Another study reported mosaicism in 1.7% of blood and buccal genomic DNA samples using a large cohort of adult individuals [76]. These numbers for mosaic mega-base range aberra-tions should be compared to ~1% of mosaicism for chromosomal aberrations described in a preselected cohort of children assessed by clinical geneticists [65]. Reports have shown that SNP arrays are able to detect mosaicism in as low as 5-7% of cells [65, 77]. In paper I [77], we showed post-zygotic ge-netic variation for the whole chromosome X in ~7% of cells. Furthermore, methods for enrichment of selected regions of the genome coupled with next generation sequencing technology have been introduced as an alternative strategy for detection of structural genetic variation [78].

Post-zygotic genetic variation in health and disease There are two well characterized examples of locus specific post-zygotic changes in the nuclear genome that occur frequently in healthy individuals. The first is occurrence of somatic rearrangements in immunoglobulin (Ig) and T cell receptor (TCR) genes in both B and T cell lymphocytes. The Ig and TCR genes are inactive in most cells and their activation is achieved by extreme shuffling of these genes in B and T cells. These processes result in production of mono-specific antibody or TCR by B or T cells, respectively [79]. The second example is variation in telomere length. Telomeres are composed of repetitive sequences at both ends of the chromosome and their length gradually shortens after each cell division. Telomere lengths possess tissue-specific loss rates and have been discussed in the context of senes-cence, aging and cancer [80-83]. Post-zygotic genetic variation has been linked to various disorders [59, 84]. Some recent instances for conditions relating to post-zygotic variation are: Proteus syndrome [85], different vascular anomalies [86] and congenital dyskeratosis [87]. Furthermore, occurrence of genetic variation in genes associated with Duchenne muscular dystrophy (DMD) and Neurofibromato-sis type 1 (NF1) are two examples that can increase the perception of post-zygotic genetic variation on the phenotype [58]. DMD is an X-linked lethal disease with the prevalence of nearly 1:3500 in male newborns [88]. Eighty seven percent of DMD cases possess structural rearrangements [89]. DMD gene contains two deletion hotspots: a major hotspot located around exons 45-52 (distal) and a minor one situated around exons 3-19 (proximal). Du-

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plication of exon 2 is the most common identified rearrangement whereas deletions of only this exon have not been described. DMD is an example where different parts of a gene display various mosaic frequencies for the structural rearrangements [90, 91]. NF1 is autosomal dominant disorder affecting 1 in 3500 individuals. The predominant type of deletions span ∼1.4 Mb on 17q (known as type I deletions) and take place due to inter-chromosomal recombination during meiosis [92, 93]. In one study [92], 40% of NF1 cases exhibited different patterns of mosaicism for microdele-tions. The percentage of cells with deletion was much higher in peripheral leukocytes than buccal smears or peripheral skin fibroblasts and the mosaic cases didn’t display the mental retardation and facial dysmorphism common-ly linked to NF1 microdeletions. They reported a new type of NF1 micro-deletions encompassing 1.2 Mb (known as type II deletions) which are me-diated by intrachromosomal recombination during mitosis.

Figure 2. Velvet twins. Oil painting by Mary Walters (1997/1998). Figure taken from Boomsma D, Busjahn A, Peltonen L. Classical twin studies and beyond. Na-ture Reviews Genetics 2002; 3(11):872-82 with permission. The figure is courtesy of Flatland Foundation, Amsterdam, the Netherlands and has been used with their permission as well.

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It has been shown that healthy subjects could carry aberrations typical for a disease. For instance, in paper II, we described subject ULSAM (Uppsala longitudinal study of adult men) case 697, who is generally healthy but car-ries a CNNLOH/aUPD on 4q in blood cells. This aberration is typical for myelodysplastic syndrome (MDS) patients but USLAM-697 doesn’t exhibit any phenotype relevant to MDS [73]. This scenario has also been shown when comparing cancer-affected and cancer-free groups in two recent publi-cations. Cancer-free cohorts exhibited majority of aberrations that were present in cancer-affected individuals but at a lower frequency [58, 74].

Twins in genetic research MonoZygotic (MZ, also called identical) (Figure 2) and DiZygotic (DZ, also called fraternal) twins are common and represent an important genetic re-source for the understanding of genetic diseases and various other pheno-types. Twin biology has been studied for decades and numerous twin-based repositories exist around the world [94]. The twinning frequency varies over time and geographic location. This variation is mostly due to DZ twinning rate since the rate for MZ twinning is fairly constant [95]. About 3% deliv-eries in the U.S. are twin births, and ~10% of these are monozygotic [96, 97]. MZ twins genetically span the boundary between two subjects. They are two different persons, but can also be treated as a single subject, genet-ically matched at the conception and present in two copies. MZ co-twins provide the best closely-matched human genetic controls possible. They are age matched; they essentially share the same environment throughout gesta-tion and usually similar environments thereafter. Most importantly, their nuclear genome is identical at conception. DZ twins on the other hand arise when two oocytes are fertilized by two different sperm cells and are inde-pendently implanted in the uterus. Two DZ twins therefore share only as much of their genomes as any two siblings would [31]. There are three subtypes of MZ twins according to placentation: 1) Dichori-onic diamniotic; 2) Monochorionic diamniotic; and 3) Monochorionic monoamniotic (Figure 3) [98]. Dichorionic diamniotic twins have separate placentas and membranes and their division occurs between 0 to 3 days after fertilization [98]. In normal embryonic development, twins with mono-chorionic diamniotic placenta divide between day 4 and day 7 after concep-tion and MZ twins with monochorionic monoamniotic placenta probably separate between day 4 and day 14 after conception [98]. Table 1 describes the data for three types of MZ twins. Conjoined twins are yet another type

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of twins, which are connected by different parts of their body. Theoretically, these type of twins arise later than 14 days after conception [98].

Figure 3. Classification of monozygotic twins according to placenta and mem-branes. A= dichorionic diamniotic pregnancy, B= Monochorionic and diamniotic pregnancy, C= monochorionic and monoamniotic pregnancy. (Figure taken from Hall JG. Twinning. The Lancet 2003;362(9385):735-43 with permission).

Table 1. Time of division for three types of MZ twins. Table taken from Hall JG. Twinning. The Lancet 2003; 362(9385):735-43 with permission).

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Traditionally, phenotypic discordance in MZ twins has been attributed to the environment and variable penetrance. The classical twin method has been applied since the 1920’s as a tool for evaluating whether a trait is influenced mainly by environmental or genetic factors. This method compares the con-cordance rates of MZ twins that are raised together with the same measures as same-sex DZ twins that are also raised together. A heritable phenotype will be more concordant in MZ twins than in DZ twins [99]. This type of analysis has been employed in studies of a large number of pheno-types/diseases, for instance, asthma, attention-deficit hyperactivity disorder (ADHD), autism, psoriasis, cancer and obesity [100-109]. Figure 4 shows selected examples of result from classical twin analysis.

Figure 4. Percentage of variances elucidated by genetic factors (purple), by shared environmental factors (green), by unique environmental influences (beige) and by differences in age (blue). The phenotypes were evaluated in Dutch female and male twins (and in some cases also in their parents and siblings). The numbers in the brackets gives the modal age of the sample in years. Phenotypes include cardiovas-cular risk factors, and personality and cognitive traits. The heritability for a behav-ioural as well as cardiovascular risk factor, such as the number of cigarettes smoked per day, is about as high as for lipoprotein(a) levels. For personality traits and indi-ces of psychopathology, heritability is ~50%, with a higher estimate for internalizing and externalizing problems in young children. Heritability of intelligence is age dependent and rises rapidly between 5 and 18 years of age. There are few differ-ences in heritability between females and males. HDL, high-density lipoprotein; LDL, low-density lipoprotein. (Figure taken from Boomsma D, Busjahn A, Pel-tonen L. Classical twin studies and beyond. Nature Reviews Genetics 2002; 3(11):872-82 with permission).

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The above briefly mentioned classical twin studies depend on several gen-eral assumptions: 1. MZ twins are 100% identical, while DZ twins share as much of their

genomes as any two siblings; 2. Scientists can reliably distinguish between MZ and DZ twins; 3. The environments of MZ and DZ twins are controlled and are approxi-

mately equal; and 4. The results from twin studies can be reproduced using the single-born

population.

A criticism of the conclusions from the classical twin studies concerns the effects caused by different environments have not been considered [110]. It has also been suggested that epigenetic differences between two MZ co-twins can result in some phenotypic discordance, e.g. in aging and schizo-phrenia [111-113].

Our research group was the first to describe that the bulk of studied MZ twin pairs had one or more within-pair differences for structural genetic rear-rangements (CNVs; see above). Hence, they are not 100% genetically iden-tical. This principle is valid for MZ twins that are both healthy concordant and discordant for a disease [37]. This indicates the possibility of targeting cohorts of phenotypically discordant MZ twins as a tool to characterize ge-netic de novo predisposition factors by uncovering differences for CNV be-tween affected and unaffected MZ twins of the same pair. The advantage offered by a MZ twin-model is that any within-pair difference must be aris-ing post-zygotically; i.e., at any time during the twins’ lifetime. Further-more, the analyses of CNV differences between MZ twins are not confound-ed by hundreds or thousands of CNVs that normally occur between two un-related subjects [14, 114]. The causes of MZ twinning in humans are unknown. It has been suggested that both genetic and epigenetic mechanism maybe associated with splitting of the early embryo [115]. The incidence rate of MZ twinning in female exhibits an excess compared to males and this frequency is more noticeable in monochorionic diamniotic MZ twins, and is even more distinguishable among monochorionic monoamniotic twins and conjoined twins, signifying an association with the timing of twinning [98, 116]. It has been suggested that the skewed X inactivation with one cell mass inactivating the paternal X chromosome and one inactivating the maternal X chromosome could de-scribe this excess [98, 116]. Other suggestion include: damage to the inner cell mass, delay in fertilization during in vitro fertilization or ovulatory drugs that could result in physical separation of the cell mass into two parts. A more probable scenario is that some blastocyst cells develop a change (muta-tion, number of chromosomes, change in gene control as in imprinting, etc.)

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leading to recognition of these cells as foreign parts by the other cell mass resulting to establishment of two separate cell masses [98].

Genetics of aging There has been a dramatic rise of human life-span; by 20-years during the second half of the 20th century. This trend is expected to continue world-wide, with an average human life-span increasing another 10 years by the year 2050 [117]. Aging has been described as a complex process of cellular senescence that leads to compromised stress response, homeostatic imbal-ance and raised risk of disease [118]. Aging is the largest risk factor for the most of common human disorders [119]. Therefore, studies of aging human cohorts may be useful for discovery of mutations that are causative for of common human disorders. A popular hypothesis behind aging and possible future treatments of age-associated conditions is the concept of “the com-pression of morbidity”. This idea implies that a healthy aging is achieved by postponing the onset of age-associated disease, by efficient treatment of age-related defects [120].

Genetic analyses of the normal aging have been inspired and dominated by search for genetic components of longevity, both in humans and various experimental models. The examination of human longevity/aging as a ge-netic trait by application of classical twin methodology shows that the inher-ited (germ-line) component of human aging is rather small. Several com-prehensive studies have been performed and the estimation of heritability behind human aging/longevity is 15-30 % [121-125]. The recent, well-powered and comprehensive genome-wide association studies (GWASs), relating single nucleotide polymorphisms (SNPs) to longevity, have provid-ed little new information. There are only three confirmed SNPs that are reproducibly associated with survival into old age and functional signifi-cance of these findings is incomplete [126-128]. Thus, there is a limited amount of data about specific inherited SNPs/other genetic variants (con-firmed in independent studies) associated with either longevity or higher mortality. A very recent and the first of its kind study relating the germ-line (inherited) structural genetic variants (copy number variation, CNV) with age/mortality suggested that the overall burden of large common deletions in 11p15.5 and 14q21.3 regions are associated with increased mortality [129]. In summary, literature suggests that factors other than inherited genetic make-up are actually dominating as determinants of the human life-span. These factors are still largely unknown and may be responsible for the re-maining 70-85% of aging-related components. The above reasoning has led to a hypothesis that among these unknown genetic factors might be an ac-

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cumulation of somatic, acquired during life, genetic changes in the genome of human cells in vivo, which is the focus of work in paper II.

Other well explored genetic component associated with human aging is mu-tations in mitochondrial DNA (mtDNA). Mutations of mtDNA cause a vari-ety of human mitochondrial diseases and are also heavily implicated in age-associated disease [130]. A hypothesis is that most of the mutations are cre-ated as replication errors during embryogenesis/early stages of life and then undergo clonal expansion in adult life [131, 132].

Environmental component behind aging is also very important. Dietary control is a major environmental factor, which has a profound effect on many aspects of health, including aging. Caloric restriction (CR) is by far the most effective environmental manipulation that can extend maximum lifespan in many species, including primates [133]. In humans, it is still not possible to firmly conclude whether CR has the ability to prolong a healthy and productive life [134], although this seems likely.

Finally, studies of epigenetic marks have also been performed in the context of aging. The CpG-methylation of DNA, preferentially clustered within CpG islands, is the best studied aspect of epigenetics in aging. Studies have convincingly shown changes in the pattern of DNA methylation in samples collected at different age. However, only a minority of subjects was studied in a longitudinal fashion [113, 135-139]. This type of analysis is neverthe-less a viable path towards defining aging-specific biomarkers. For instance, as monozygotic twins are ideal controls for each other (identical at concep-tion), they represents a good model for studying somatic epigenetic changes linked to aging. In the first study of monozygotic twins varying in age from 3-74 years, global and locus-specific epigenetic differences between the pairs were found to increase with age [113].

Blood cells, immune system and genotyping Hematopoietic stem cells generate different blood cells. These self-renewing cells can produce either myeloid or lymphoid progenitor cells. Progenitor cells don’t possess the self-renewal properties [140]. Subse-quently, myeloid progenitor cells generate granulocyte-monocyte, eosino-phil, basophil and erythroid progenitor cells. Lymphoid progenitor cells produce natural killer (NK) cells as well as T- and B-cell progenitors (Figure 5). White blood cells or leukocytes are cells from the immune system and are divided into: 1) Granulocytes or polymorphonuclear leukocytes, which include neutrophils, basophils and eosinophils; and 2) Agranulocytes or

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mononuclear leukocytes which consist of lymphocytes, monocytes and mac-rophages. Different blood cells present various cluster of differentiation (CD) on their surfaces. For instance, CD4 is used as a marker for T helper cells (Th cells) (CD4+) and CD19 is expressed on the surface of B lympho-cytes (CD19+) during their development [141].

Figure 5. Generation of blood cells by hematopoietic stem cell. Figure modified from the book “Kuby Immunology” by Kindt, T.J. et al., sixth edition, W. H. Free-man and Company, 2007 with permission.

Both B and T lymphocytes are parts of the immune system and play essential roles in the immunological responses. B cells arise in bone marrow and produce antibodies once encountering an antigen. T cells pass functional maturity in the thymus and perform cell-mediated immune responses [142].

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Both B and T cells recognize and react to antigens by means of cell surface proteins known as antigen receptors. During development of lymphocytes, millions of diverse clones of cells are formed each possessing a unique spec-ificity for antigen receptor. At this stage, cells that react with the antigens inside body are omitted in a process known as immune tolerance. Upon introduction of a foreign antigen, the cells with a specific recognizing anti-gen receptor are expanded in a process known as clonal selection [142]. Combined immunodeficiencies for B- and T-cells could result in severe combined immunodeficiency (SCID) disorders [143]. In T-B- SCID, both T and B cells are absent. Various diseases are associated with this group in-cluding Artemis deficiency and reticular dysgenesis (aleukocytosis). Reticu-lar dysgenesis is a very infrequent autosomal-recessive SCID, which com-monly results in very early death. Reticular dysgenesis is the most severe form of inborn SCID and is associated with a dramatic reduction in the num-bers of B and T cells, thrombocytes and granulocytes due to impaired matu-ration of both lymphoid and myeloid precursor cells. This disease is caused by mutations in the gene encoding the mitochondrial energy metabolism enzyme adenylate kinase 2 (AK2) [143, 144]. In T-B+ SCID, T cells are absent and generated B cells are defective but may exhibit an increase in the number. Other types of SCIDs have been described [143].

Whole blood is the primary source of DNA in GWASs as well as in other genetic studies and it is usually assumed that whole blood DNA reflects the same genomic profiles for all blood cells. In paper II [73], we showed that sorted blood cells can display different genomic profiles and different sorted blood cells exhibit various degrees of aberrations. Another recent report [145], indicated that there is a variation in the methylation profile of sorted blood cells. These two studies point to the importance of both genomic and methylation studies on sorted blood cells instead of only whole blood DNA and emphasize that drawing conclusion just based upon results from whole blood DNA could result in misinterpretation of the data.

Encyclopedia of DNA Elements (ENCODE) After completion of the human genome project in 2003, the ENCODE pro-ject [146] emerged as one of the most important projects in human genetics, which focused on identification of all the functional elements in the human genome sequence. The ENCODE project started in 2003 and was launched by the National Human Genome Research Institute. The project commenced in three phases: a pilot phase, a technology development phase and a produc-tion phase [146]. The pilot phase focused on analysis of functional data on 1% (~30 Mb) of the human genome [147]. This 30 Mb was distributed in 44

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genomic regions. Nearly, 15 Mb were located in 14 regions for which sig-nificant amount of biological knowledge were available. The other 15 Mb were positioned in 30 genomic regions chosen by a stratified random-sampling strategy [147]. In the pilot phase, a consortium of 35 groups mapped a variation of sequence elements comprising genes, exons, promot-ers, enhancers, repressor or silencer sequences, sites of origin and termina-tion of replication, methylation sites, transcription factor binding sites, RNA transcripts, DNase I hypersensitive locations, chromatin modifications and conserved sequences [148]. The result from the pilot phase indicated that the human genome is transcribed pervasively and led to identification of multiple previously unknown transcription start sites as well as various novel non-protein-coding transcripts [147]. The production phase of the ENCODE project was initiated in 2007 and targeted the entire genome. The main as-says used in the ENCODE project are chromatin immunoprecipitation se-quencing (ChIP-seq), RNA sequencing (RNA-seq), DNase I hypersensitivity and DNA methylation [149]. An extensive set of results relating to experi-ments including 147 different cell types were published in 2012 [149]. These results indicate that 80.4% of the human genome contributes to at least one biochemical RNA- and/or chromatin-associated event in at least one cell type especially outside of the well-characterized protein-coding sites [149].

Interferons and interferon receptors In paper III, we show post-zygotic genetic variation for a VNTR in a puta-tive enhancer element of the locus between the IL10Rβ and IFNAR1 genes on chromosome 21q22.11. This variation might influence the transcription of the genes located in the vicinity of this putative regulatory element. Here, interferons, their involvement in disease including cancer and their signifi-cance in disease treatment are introduced.

Interferons (IFNs) is a group of cytokines that bind to receptors on the cell surface of the target cells and induce variation in gene expression leading to an antiviral state or other cellular responses important in the immune re-sponse [150]. Interferons play an important role in the innate immune re-sponse to viral infections [151]. Intereferons are categorized into type I, type II and type III based upon receptor specificity and sequence similarity [152]. Interferon alpha (IFN-α) and interferon beta (IFN-β) are examples of type I interferons [152-154]. IFN-α proteins are produced by leukocytes and increase resistance of cells to viral infection [155]. IFN-β is produced by fibroblasts [156]. Interferon alpha, beta and omega receptor (IFNAR) is the receptor for type I interferons and comprises two components: interferon

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alpha, beta and omega receptor 1 (IFNAR1) and interferon alpha, beta and omega receptor 2 (IFNAR2) [154]. Interferon gamma (IFN-γ) is the sole type II IFN [152] and is produced by T cell lymphocytes [156]. IFN-γ acti-vates macrophages and increase expression of major histocompatibility complex (MHC) proteins [155]. IFN-γ receptors consist of interferon gam-ma receptor 1 (IFNGR1) and interferon gamma receptor 2 (IFNGR2) [154]. Type III interferons include interferon lambda 1 (IFN-λ1), interferon lambda 2 (IFN-λ2) and interferon lambda 3 (IFN-λ3) [153]. Type III IFN receptors consist of interferon lambda receptor 1 (IFN-λR1) and interleukin 10 recep-tor beta (IL10Rβ) [153]. IFNAR1, IFNAR2, IFNGR2 and IL10Rβ are located in a cytokine receptor gene cluster on human chromosome 21q22.1 [154]. IFNs and their signaling pathways have been linked to various disease-related processes. For instance, type-I IFN (IFN-α)-induced signaling re-duces in both T and B cells in 3 groups of cancer patients including breast cancer, melanoma and gastrointestinal cancer. Type-II IFN (IFN-γ)-induced signaling reduces in B cells of these cancer patient groups [157]. Further-more, type-III IFN-induced apoptosis in human lung cancer cells has been reported [158]. IFN-α has been used extensively as a therapeutic agent [159]. IFN-α has been applied as an approved treatment methods against chronic hepatitis C virus (HCV) infections for two decades [156, 160]. IFN-α is also applied as a treatment method for infection caused by human herpesvirus 8 known as the aetiological agent in Kaposi’s sarcoma (KS). KS is a formerly infrequent form of cancer, which is associated with acquired immunodeficiency syn-drome (AIDS) [156, 161]. Tumors caused by infection with human papillo-matosis virus (HPV) group such as recurrent respiratory papillomatosis [162] and genital warts [163] have been treated with interferons [156]. Further-more, juvenile laryngeal papillomatosis has been treated with IFN-α [164]. In treatment of renal-cell carcinoma, about 14% of metastatic renal-cell car-cinoma cases respond to therapy with IFN-α alone and this agent has been used in conjunction with interleukin-2 [165]. Treatment with interferon is applied as adjuvant therapy in cutaneous melanoma that has metastasized to local lymph nodes [166]. Anti-angiogenic characteristic of interferons has made them applicable in treatment of haemangiomas (benign tumors of blood vessels) in infants when usual corticosteroid therapy is not operational [167]. Intramuscular treatment with IFN-β decreases the annual rate of re-lapses in multiple sclerosis (MS) patients [168]. Application of natalizumab, a recombinant monoclonal antibody to an integrin, reduces the rate of MS relapses by 50% when compared with treatment with only IFN-β [169]. Anti-IFN-γ therapy have been applied in treatment of various autoimmune diseases comprising rheumatoid arthritis and MS as well as different auto-immune skin diseases such as psoriasis [170].

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Methods

Array comparative genomic hybridization (aCGH) Array-CGH is a frequently used method for detection of unbalanced struc-tural variation such as CNVs. This method has emerged from conventional metaphase-CGH, in which mixture of differentially labeled test (usually tumor sample) and reference DNA (control sample) are competitively hy-bridized to normal metaphase chromosome spreads on a glass slide [171]. In aCGH, chromosomes are replaced by large number of target sequences with known chromosomal position spotted onto a glass slide, providing a higher resolution as compared to metaphase-CGH. Arrays can be made of genomic clones, cDNA, PCR fragments or oligonucleotides [172-174]. In aCGH, test and reference DNA are labeled with two different dyes and co-hybridized to a microarray. Then, the array is scanned and relative fluorescence intensities are indicative of DNA copy number difference (Figure 6) [171]. aCGH is unable to detect polyploidy, balanced inversions and balanced translocations as well as copy number neutral loss of heterozygosity (CNNLOH) [171, 175].

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Figure 6. Procedure of array CGH. To block the repetitive sequences, fluorescently labeled test and reference DNA are mixed with the Cot-1 DNA. After hybridization to microarray, aberrant chromosomal regions can be distinguished based upon the difference in relative fluorescence intensities.

Illumina single nucleotide polymorphism genotyping Single nucleotide polymorphism (SNP) arrays are widely used in genome-wide association studies (GWASs) and usually provide a higher resolution to study structural variation compared to aCGH. Ilumina SNP arrays use the Infinium whole genome genotyping (WGG) procedure. Two Infinium as-says were introduced: Infinium I or WGG-allele-specific primer extension

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A B

CD

(ASPE) [176] and Infinium II or WGG-single-base extension (SBE) [177], which is dominating among Illumina products. WGG consists of four steps: 1) Whole genome amplification (WGA), 2) Array-based hybridization of gDNA to a/two 50-base capture probe(s) depending on ASPE or SBE, 3) array-based SNP scoring and 4) signal amplification [176, 177]. WGA is carried out using multiple displacement amplification (MDA) method [178]. SBE has a more robust chemistry than ASPE, since SBE is an “end-point” while ASPE is a “kinetic” assay. In ASPE, two beads for each SNP are re-quired and one single color scores the SNPs while in SBE only one bead type is used and SNP scoring uses a two-color/hapten format [176, 177]. Haptens are small molecules that can bind covalently to a carrier protein to create an immune response and can also bind to an antibody [179]. In SBE, haptens are detected by a two-color antibody-based method which is fol-lowed by signal amplification [177]. Next, the fluorophores are excited, the image intensities extracted and the generated data are evaluated to determine SNP genotypes using Beadstudio/GenomeStudio softwares from Illumina company (Figure 7).

Figure 7. An Illustration for procedure of Illumina SNP genotyping according to Infinium II assay. In panel A, the grey part at the bottom displays the chip onto which the beads (grey circles) are immobilized and probe-A sequence (grey lines) are attached to them. The beads are coated with multiple copies of an oligonucleo-tide probes that target specific locus in the genome. Genomic DNA (blue lines) is captured by the probes on the bead stopping one base before locus of interest. In Panel B, allele specificity is conferred by a single base extension (SBE) that incor-

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porates one of four nucleotides. The dideoxynucleotides are labeled with 2,4-dinitrophenol (DNP) or biotin as haptens. In Panel C, the haptens are recognized by fluorescent-labeled antibodies using immunohistochemical sandwich assay. In panel D, after laser excitation, the labeled nucleotide emits a signal which is detected by Illumina scanner. Intensity values for each color convey information about the allel-ic ratio of a given locus. For example, if the color representing “A” is approximate-ly as intense as the color representing “G”, the genotype at that locus is heterozy-gous. For simplicity, the allelic ratios were displayed on the beads. The figures are courtesy of Illumina Inc. and were used with permission from Illumina Inc.

Illumina SNP genotyping consists of two main data tracks: Log R ratio (LRR) and B allele frequency (BAF). For each probe on the SNP array, LRR is the logged ratio of observed to expected signal intensity. In the nor-mal state, LRR value is zero and any deviations from zero indicate copy number change. BAF is the proportion of hybridized sample that carries the B allele as designed by the Illumina Infinium assay. In the normal state, heterozygous SNPs have BAF value of 0.5 while homozygous SNPs display BAF value of 0 or 1 depending on the allele type and any deviations from these values are indicative of structural aberrations [180]. These two tracks provide a great tool for detection of de novo somatic copy number changes. Different algorithms for identification of copy number variants from SNP arrays were developed. Comparison between these algorithms showed that Nexus Rank and SNPRank display low specificity and high power, Nexus Rank oversizes CNVs and PennCNV identifies one of the fewest numbers of CNVs [181]. In the three described papers in this thesis, we analyzed Illu-mina output files by using Nexus Copy Number version 5.1 (BioDiscovery, CA, USA), which applies a ‘‘Rank Segmentation’’ algorithm based on the circular binary segmentation (CBS) approach [182]. We used ‘‘SNPRank Segmentation’’, which is a very similar algorithm to ‘‘Rank Segmentation’’ but utilizes BAF values in the segmentation process, producing both copy-number and allelic-event calls.

SNP arrays allow for evaluation of genotypic concordance between MZ twins of a pair or between different samples (e.g. from different tissues) de-rived from the same subject. The expected value for different tissues from the same person should be as genotypic concordance for MZ twins (> 99.9% identical genotypes). Unlike aCGH, SNP arrays are able to detect polyploi-dy and CNNLOH but are unable to identify balanced inversions and translo-cations [171, 175].

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Quantification of percentage of aberrant cells carrying structural variation Somatic disorders can arise only if a sufficient proportion of cells are affect-ed by aberration. This indicates the necessity of application of alternative methods to measure the percentage of aberrant cells. Various methods have been developed to quantify the percentage of aberrant cells which carry structural variation. Here, I briefly present 3 methods: 1) Fluorescent in situ hybridization (FISH); 2) quantitative real-time polymerase chain reaction (qPCR); and 3) mosaic alteration detection (MAD) which utilizes Illumina SNP array data.

Fluorescence in situ hybridization (FISH) FISH is a molecular cytogenetic method used for visualization of specific labeled DNA probes hybridized to chromosomal structures. FISH is broadly used today as a diagnostic method to detect numerical and structural abnor-malities in the DNA. This technique can identify genetic alterations such as deletions, translocations, inversions, and gene amplifications. However, certain genetic rearrangements such as insertions or smaller aberrations may be difficult to detect using FISH [183, 184]. FISH can be performed on in-terphase nuclei or metaphase chromosomes. In summary, the target DNA is attached to a microscope slide. The specific DNA probe which is labeled by a hapten or a fluorophore and the target DNA are denatured to yield single-stranded DNA. The specific DNA probe is hybridized to its complementary sequence in the target DNA and the signal is detected using a fluorescence microscopy. Application of probes labeled with different fluorophores allow for detection of various target sequences [171]. In calculation of the per-centage of aberrant cells, two probe sets are used. One probe set is comple-mentary to a normal segment of an individual’s genome while the other set binds to an aberrant segment of the genome. Application of sufficient num-ber of probes in each set allow for calculation of the percentage of cells with aberration.

Quantitative real-time polymerase chain reaction (qPCR) Quantitative real-time PCR is a widely used method in confirmation of ex-pression analysis data and copy number variation in the genomic DNA (gDNA). It is also possible to measure the percentage of cells that carry a

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copy number change with qPCR. To do so, two ultra-conserved elements (UCE) in the genome are used as controls, which show no known natural variation in the human population [185]. Prior to qPCR, amplification effi-ciency of all the primer pairs should be evaluated. During qPCR, gDNA is amplified with three different primer pairs. Two primers pairs amplify two separate UCEs while the third primer pair targets the region with the sus-pected copy number change. The average cycle threshold (Ct) value of one UCE is used to normalize the average Ct values of the other UCE and the test locus. Next, the normalized Ct values are used to calculate the copy-number ratio for the UCE and the test locus [73, 186, 187]. In measurement of subtle changes, to enhance the statistical power, qPCR experiments should be replicated.

Mosaic alteration detection (MAD) Mosaic alteration detection (MAD) is a software that is effective to detect allelic genomic imbalances that are smaller in size (~500 Kb) and/or affect-ing a low percentage of cells. This method utilizes both LRR and BAF of the samples that were genotyped with Illumina SNP arrays. The software detects abnormalities by calculation of the B deviation (Bdev), which is the deviation of the BAF signal from the expected value of 0.5 for heterozygous in a normal state. Once a segment is called by assessing BAF value, the average LRR of the segment is calculated to determine whether the aberra-tion is affecting the copy number [188]. In paper II, we used the MAD pro-gram [76] for the calculation of the number of cells affected by deletions, gains and CNNLOHs.

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Present investigations

Aims

• Paper I: To describe genetic variation for the chromosomes X and Y in a pair of healthy monozygotic twins using Illumina SNP geno-typing and FISH.

• Paper II:

1) To show that there exist accumulation of post-zygotic, ac-quired during lifetime genetic changes in the nuclear genome of hu-man blood cells in vivo;

2) To show the dynamic changes of large- and small-scale post-zygotic genetic variation;

3) To compare the genetic profiles of various types of sorted blood cells versus the whole blood DNA.

• Paper III: To characterize post-zygotic genetic variation in a VNTR-based putative enhancer locus between IL10Rβ and IFNAR1 genes.

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Paper I. Somatic mosaicism for chromosome X and Y aneuploidies in monozygotic twins heterozygous for sickle cell disease mutation

Introduction MZ twins are used in studying diseases, evaluating quantitative trait loci, estimating heritability, identifying differences in gene expression, and exam-ining hypotheses regarding gene-environment interactions [94]. Thus, it is important to determine “the normal baseline” (the distribution in the genome and the frequency) of CNVs between any twins from unselected MZ pairs. Mosaic aneuploidy is one of the best studied types of mosaicism due to ap-plication of cytogenetic and molecular analyses in the routine diagnosis of patients referred to clinical geneticists. Aneuploidy involving chromosome X (usually monosomy) is one of the predominant aberrations [189-191]. There are reports showing MZ twins discordant for phenotypic sex and vari-able degree of 45,X/46,XY mosaicism [192, 193]. Furthermore, MZ twins discordant for Ullrich-Turner syndrome and varying degree of mosaicism for 45,X/46,XX have also been reported [194].

Results and discussion We genotyped blood DNA of 55-year-old female MZ twins with sickle cell trait (STr) using Illumina human 610 SNP array. Complete physical exami-nation showed essentially normal phenotype. Both twins only revealed moderately high blood pressure controlled by treatment. Both twins had children, healthy siblings and healthy parents. The twins have never re-ceived blood transfusion or bone marrow transplantation. Illumina SNP genotyping indicated that the studied twins are monozygotic. All 14,976 SNPs from chromosome X that were scored in both twins showed identical genotypes. SNP genotyping revealed a copy number difference for chromo-some X in a proportion of cells in twin 1. Interphase Fluorescence in situ hybridization (FISH), confirmed monosomy X (45,X) in 7.2% of nucleated blood cells. Unexpectedly, FISH analysis detected 45,X and 46,XY lineages each present in 1.1% of cells in twin 2.

The probable mechanism behind the aneuploidies is an error in the meiotic division of either the father or the mother. 24,XY gamete meeting 23,X gamete, alternatively 24,XX and 23,Y gametes joined together. This has been followed by trisomy rescue in the mitotic divisions of the embryo, giv-ing rise to one normal predominant 46, XX cell lineage and two less preva-

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lent abnormal lineages (45,X and 46,XY). The latter lines were distributed with different frequencies to the twins. As samples from the parents were not available, we cannot resolve the question of origin of the abnormal gam-ete. The SNP genotyping showed 5,056 heterozygous chromosome X SNPs in both twins. This indicates that two different X chromosomes from both parents were present in the zygote and the mechanism behind our results is incompatible with a normal 46,XY zygote affected by mitotic non-disjunction events leading to mosaic aneuploidies. The presence of 46,XY cells in only one twin shows that bone marrow invasion from one MZ twin to another is unlikely to be responsible for the observed picture, since in this case twin 1 should also contain 46,XY cell lineage. Our results illustrate a considerable plasticity of the human genome for tol-erating large CNVs in healthy subjects. Our findings add to delineation of the frequency of structural genetic variation in normal MZ twins and could help to determine the genomic distribution and frequency (or “baseline”) of CNV between MZ twins.

Paper II. Age-related somatic structural changes in the nuclear genome of human blood cells

Introduction Apart from premature aging syndromes [195], several comprehensive studies have been performed and estimated that the heritability behind normal hu-man aging/longevity is 15-30% [121-125]. Therefore, 70-85% of aging-associated factors are not heritable and at least a part of the explanation might be due to somatic, acquired during life, genetic changes in the genome of various lineages of human somatic cells.

There are indications that chromosomal remodeling in the nuclear and mito-chondrial genomes increases with age [113, 196-200]. Mutations in mito-chondrial DNA has been associated with human aging and it has been shown that these mutations cause a variety of human mitochondrial diseases and are also heavily implicated in age-associated disease [130]. Reports described frequency of 1.7% for large-scale post-zygotic mosaicism for large-scale aberrations [76]. Reports from our group describe that adult monozygotic (MZ) twins and fully differentiated human tissues frequently display somatic CNVs [37, 38]. In spite of these studies, little is still known about the rate of

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formation and distribution of de novo post-zygotic somatic CNVs in normal cells and whether these aberrations accumulate with age. So, we hypothe-sized that the nuclear genome of blood cells in vivo might accumulate CNVs with age and applied age-stratified MZ twins as a starting point to test this hypothesis. We followed these twin-based analyses using cohort of single-born elderly subjects from the ULSAM study.

Figure 8. The whole genome profiles in longitudinal analysis of 4 peripheral blood samples collected from subject ULSAM-697 at the ages of 71, 82, 88, and 90 years (panels A, B, C and D, respectively). This figure illustrates a clonal cell expansion containing a terminal CNNLOH/aUPD encompassing 103 Mb of the long arm of chromosome 4, with an increase and a decline in the proportion of affected cells at different ages. Each panel consists of images from Illumina SNP array displaying the BAF-values, as CNNLOH/aUPD is not detectable using LRR data. The estimat-ed percentage of cells displaying CNNLOH/aUPD on chromosome 4 is shown for each sample. This aberration was not detectable at the age of 71, reached approxi-mately 58% at the ages of 82 and 88 years and declined radically to about 30% of cells at the age of 90 years. Also, this figure shows the BAF-profiles for the whole

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genome from genotyping of sorted blood cells (CD19+ B lymphocytes, CD4+ T lymphocytes, and granulocytes) as well as skin fibroblasts collected at the age of 90 years (panels E, F, G and H, respectively). Sorting of blood cells at the age of 90 years showed that CD4+ T-cells and granulocytes were affected to a similar degree, as identified in DNA from unsorted blood at the same age. However, CD19+ B-cells were unexpectedly not affected by this aberration. Thus, both myeloid and lymphoid lineages were affected to a similar degree, with the notable exception of B-lymphocytes. Panels I and J show statistical analysis of data. Panel I shows comparisons of “BAF-value deviation from 0.5” for heterozygous probes only and within the aberrant region of 4q, derived from analysis displayed in panels A through D. Similar analysis is shown in panel J for data derived from panels E through H. The proportion of cells with the 4q aberration changes with time and between different types of cells. These changes are significantly different between all samplings (ANOVA p<0.001; Tukey's test for multiple comparisons). (Figure taken from Forsberg LA, Absher D, Dumanski JP. Non-heritable genetics of human disease: spotlight on post-zygotic genetic variation acquired during lifetime. Journal of medical genetics 2013;50(1):1-10, with permission.).

Results and discussion We used whole blood DNA from 318 MZ twins and 296 single-born sub-jects. 78 MZ twin pairs and 108 single-born individuals were ≥60 years old. 81 MZ pairs and 180 single-born subjects were ≤55 years old. Using differ-ent Illumina SNP beachchips, we detected megabase-range aberrations in 3.4% of subjects ≥60 years old but not in individuals ≤55 years old. Statisti-cal test supports age-related accumulation of large structural variants in the older group (Fisher’s exact test; p value = 0.00052) (Table 2).

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Table 2. Summary of megabase-range genetic aberrations. Illumian SNP genotyp-ing on whole blood DNA detected megabase-range genetic aberrations in nine out of 264 subjects ≥60 years old while none of the subjects ≤55 years old showed such aberrations. Fisher’s exact test indicate the there is a significant difference between these two age groups. (Table reproduced from paper II: Forsberg, L.A., age-related somatic structural changes in the nuclear genome of human blood cells. American journal of human genetics 2012; 90(2):217-28).

Among the studied subjects, we identified 3 healthy individuals that have aberrations characteristic of MDS and none of them were diagnosed with MDS, and their cases might represent an indolent, subclinical form of MDS. Large-scale aberrations were confirmed using Nimblegen 720K array-CGH. We used deviations in BAF as the main detection tool for small-scale struc-tural aberrations because it can detect mosaicism in as low as 5%-7% of cells [65, 77] and allow detection of deletions and duplications as well as CNNLOH. Using deviations in BAF values alone and BAF together with LRR in MZ twins, we detected 827 CNV calls at 378 loci in 87 MZ pairs. Based on frequency and/or location of these loci in the vicinity of known genes, we selected 138 loci for validation using Nimblegen 135K custom-made tiling-path array. In total, 52 of the 138 loci (38%) were validated. The validated small-scale variations were recurrent among different MZ twins. Five of these fifty-two loci were further validated with qPCR. Using SNP array data as primary detection tool and Nimblegen tiling-path array and qPCR as validation methods, we showed age-related accumulation of small somatic structural rearrangements in the nuclear genome of human blood cells.

Longitudinal analysis of individuals with structural aberrations is another interesting aspect of this study. For instance, subject ULSAM-340 contained a 20q deletion, which was barely detectable at the age of 71. The number of cells containing the 20q deletion was estimated by analysis with the MAD (Mosaic Alteration Detection) program [76, 188] to be 50.7% when this subject was 75 years old and to be 36.1% at 88 years. Another example for longitudinal analysis of individuals with structural aberrations is subject ULSAM-697 which was studied at four time points and showed a pro-

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nounced increase and decrease in the number of cells with CNNLOH on 4q. This aberration was not detectable at the age of 71, reached 58.4% at the age of 88, and declined radically to 29.9% of cells at the age of 90. When this subject was 90 years old, we genotyped sorted CD4+ cells, CD19+ cells, granulocytes, and fibroblasts, in addition to whole-blood DNA. CD4+ cells, granulocytes, and whole blood showed similar levels of aberrant cells, whereas CD19+ cells and fibroblasts appeared normal. We performed all experiments on samples taken when ULSAM-697 was 90 years old in dupli-cate with different types of arrays. Thus, in this subject, both lymphoid and myeloid cells were affected, except for, quite surprisingly, CD19+ B cells (Figure 8). Overall, the analyses performed on ULSAM-340 and ULSAM-697 suggest that the cells with aberrations have higher proliferative potential than do other cells in the immune system, but they are not immortalized be-cause they apparently disappear from circulation. These results also suggest an “autocorrection” of the immune system, given that the aberrant clones are apparently disappearing from circulation. In addition to MAD analysis, we also used qPCR to measure the percentage of aberrant cells in different sub-jects in this study. The CNNLOH/aUPD on 4q in ULSAM-697 is a typical aberration for indi-viduals affected with MDS, however this subject is healthy and has no phe-notypic signs of MDS. This aspect has also been demonstrated in compari-son of cancer-affected and cancer-free subjects. Cancer-free individuals possessed majority of aberrations that were present in cancer-affected sub-jects but at a lower frequency [74].

The somatic structural variation described in this study, probably represent only part of all the somatic changes that actually occurred in the studied cohorts because balanced inversions and translocations escape our detection and because we interrogated a fraction of all the nucleotides in the genome. Nevetheless, the chromosomal regions and loci affected in a recurrent fash-ion are candidates for containing common and redundant age-related defects in human blood cells. These mutations are presumed to provide the affected cells with a mild proliferative advantage without transforming the affected cells into immortalized cancer clones. However, the proliferative advantage for a limited number of cells will most likely affect the overall complexity of cell clones present in blood and should therefore be discussed in the context of immunosenescence, which involves loss of complexity of cell clones in both B and T cell lineages [201, 202]. We anticipate that extension of our results will allow determination of the genetic age of different somatic-cell lineages and estimation of possible individual differences between genetic and chronological age. Our findings were recently supported and extended in two other reports, which also point towards accumulation of genetic aber-

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rations with age [74, 75]. In one study, they reported that the frequency of cancer-free individuals exhibiting detectable clonal post-zygotic mosaicism events increased with age. This frequency was 0.23% for individuals under 50 years old and increased to 1.91% for those between the ages of 75 and 79, but no increase in mosaic abnormalities for individuals under age 40 were observed [74]. In the other study, the frequency of clonal somatic mosai-cism was <0.5% in subjects less than 50 years of age but increased with age to 2.7% in subjects over 80 [75]. Our results show that both large- and small-scale copy number variations are accumulating with age in the nuclear genome of human blood cells. Longitudinal analyses suggest considerable variation in the rate of accumulation of clones carrying structural changes. Furthermore, the longitudinal analysis of individuals with structural aberra-tions suggests that there is a natural self-removal of aberrant cell clones from peripheral blood.

Paper III. Post-zygotic and inter-individual structural genetic variation in a putative enhancer element of the locus between the IL10Rβ and IFNAR1 genes

Introduction The Encyclopedia of DNA Elements (ENCODE) project recently character-ized regions of transcription, transcription factor association, chromatin structure and histone modification. They used different assays including chromatin immunoprecipitation sequencing (ChIP-seq), RNA sequencing (RNA-seq), DNase I hypersensitivity and DNA methylation on 147 different cell types. Results indicate that up to 80% of the human genome contributes to at least one biochemical RNA- and/or chromatin-associated event in at least one cell type especially outside of the well-characterized protein-coding sites [149]. It has been shown that tandemly repeated sequence motifs can affect human phenotype. For example, different disorders have been associ-ated with specific unstable repeat expansions within genes. Studies regard-ing tri-nucleotide repeat expansions in the context of somatic variation have shown that CTG repeat in the 3’ UTR of the dystrophia myotonica 1 protein kinase (DM1PK) gene is larger in skeletal muscle than blood DNA in dys-trophia myotonica 1 (DM1) patients [203]. Size heterogeneity of this repeat increases also with age in the blood DNA of these patients [204]. Moreover, the age-dependent and tissue-specific somatic mosaicism for this tri-nucleotide repeat has been reported in the mouse model of DM1 [205]. In

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paper III, we present somatic genetic variation in a VNTR-locus, which has sequence properties of an enhancer.

Results and discussion Initially, SNP genotyping of 63 MZ twin pairs and multiple tissues from patients diagnosed with breast cancer suggested a frequent somatic mosai-cism in the region between IL10Rβ and IFNAR1 genes, ~4 kb upstream of the IFNAR1 gene on chromosome 21q22.11. We used whole blood DNA from 63 MZ twins and DNA from multiple tissues (peripheral blood, prima-ry breast tumor, and healthy morphologically normal breast tissue from the same breast affected with primary tumor) from 21 breast cancer patients. Each MZ twin pair and each breast cancer patient were treated as a single case. Twenty eight out of 63 MZ twin pairs (44%) and 13 out of 21 (61.9%) breast cancer patients showed this variation. The NCBI reference sequence (build 36.3) displayed a 32 bp core motif re-peated seven times in the center of the tandem repeat locus and this core was flanked by several similar motifs. We selected 17 cases for validation where Illumina results suggested somatic variation. We approached validation of variation by PCR amplification from gDNA followed by sub-cloning of the products into plasmids, preparation of plasmid DNA from up to 50 bacterial clones for each PCR reaction, analysis of insert sizes on agarose gels and Sanger sequencing using plasmid DNA as template. For alignment of se-quencing results, we used anchor 1 (A1) and anchor 2 (A2) sequences, which were non-repetitive and located close to the cassette of tandem repeat motifs. The results from Sanger sequencing confirmed the variation and established eight fully sequenced hypervariable region (HVR)-alleles, with at least two high-quality forward and reverse sequence reads, showing suffi-cient overlap to assure the correct assembly across multiple similar sequence motifs. However, the limits of current sequencing technology did not permit establishing of the full-length sequence for the five additional longer HVR alleles (based on the same stringent quality control of sequence reads that are aligned between anchors 1 and 2 segments) and their sizes were therefore estimated from agarose gels. We validated a relatively high percentage (10 out of 17; 58.8%) of cases that were suspected to contain somatic genetic variation in the studied locus from Illumina data. Further analyses indicated strong clustering of transcription factor binding site within this HVR, suggesting an enhancer. Analyses also showed the presence of a CpG island just upstream the predicted enhancer element. One of the first described and well-studied VNTR-based enhancers in the human genome is located upstream of the human insulin gene (INS) on chromosome

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11p15. This VNTR has been shown to regulate not only the expression of the closest gene (INS), but also the IGF2 gene, located downstream on the same DNA strand from INS, thus demonstrating the long range effect of the VNTR on gene transcription [48, 206]. Enhancer elements can activate gene expression independent of their orientation and are commonly scattered across large noncoding intervals, in extreme cases functioning at a distance of >1 Mb from their gene promoter target [207, 208]. Considering these points, it is reasonable to hypothesize that our proposed VNTR-based puta-tive enhancer might influence on the transcriptional regulation of neighbor-ing cytokine receptor genes (IFNAR2, IL10Rβ, IFNAR1 and IFNGR2) and the pathways they are involved in. We reported somatic genetic variation for a VNTR-based locus between IL10Rβ and IFNAR1 genes which showed both inter- and intra-individual variation. In this HVR, we found strong clustering of transcription factor binding sites, suggesting an enhancer. The identified HVR might function in transcriptional regulation of neighboring cytokine receptor genes and the pathways they are involved in. The extension of our work may unveil the normal function of this VNTR-locus in transcriptional control of genes and its possible involvement in various immune system dysfunctions, which are attributed to these cytokine receptor pathways.

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Concluding remarks and future perspectives

Post-zygotic genetic variation represents a growing field in human genetic research and has already been linked to different disorders, not necessarily limited to cancer. The presented three studies highlight the importance of research on post-zygotic or somatic mosaicism in human genetics. We em-phasize that biobanks should consider longitudinal sampling of multiple tissues from an individual to better address mosaicism in the genetic studies. Our results point to several possible future lines of investigation:

• There is a need to better define the normal baseline (genomic distri-bution and frequency) of CNVs that occur post-zygotically as changes acquired during lifetime in cohorts of healthy subjects. This can be achieved by studies of larger cohorts of monozygotic twins or adult and aging people where samples have been collected in a lon-gitudinal fashion.

• Monozygotic twin-based research in the context of post-zygotic ge-

netic variation can also be a promising tool for delineation of genetic aberrations that are linked to a specific phenotype. Cohorts of monozygotic twins that are phenotypically discordant can be used for this purpose.

• In paper II, we described chromosomal regions displaying aberra-

tions in a recurrent fashion and hypothesized that these regions are candidates for common age-related defects in blood cells in vivo. The proposed post-zygotically acquired and recurrent aberrations should be further investigated using larger cohorts of subjects, as this may reveal changes related to shorter or longer survival of aging people. This issue is closely related to a possibility of determination of the genetic age for different somatic-cell lineages and estimation of possible individual differences between genetic and chronological age.

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• We used blood cells as the main source of DNA for our analyses of post-zygotic variation. Similar studies, using DNA from other so-matic tissues (other than blood), could delineate whether there exist differences in the frequency and genomic distribution of post-zygotic CNVs between different lineages of somatic cells.

• Our results from paper II also suggest that different clones of cells

affected with post-zygotic acquired genetic aberrations have a higher proliferative potential, compared to other cell clones in blood and are therefore detectable with our method. However, although the aberrant cells likely have a higher proliferative potential, they are not immortalized and disappear from circulation. This suggests that there exist an auto-correction mechanism, probably related to normal apoptosis. Further investigations should focus on characterization of the dynamics of these clonal cell expansions as well as which specif-ic genes are mutated in these expanding-contracting cell clones.

• In paper III, we showed post-zygotic genetic variation in the locus

between IL10Rβ and IFNAR1 genes. Further characterization, could reveal the influence of the proposed variation on expression pattern of the genes located in the vicinity and their pathways in the aberrant cells compare to normal cells. Interferons, acting via multiple recep-tor genes encoded from the locus studied by us, are already used in clinical treatment of patients with various conditions. Our study may allow for future analysis aiming at understanding of why there often is a variable response to treatment with interferons.

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Acknowledgements

First of all, I would like to thank my opponent, Associate Prof. David Gis-selsson Nord, and the committee board, Prof. Caroline Graff, Associate Prof. Cilla Söderhäll, and Associate Prof. Larry Mansouri for reviewing my thesis. I would like to express my warmest gratitude to several people: My supervisor, Jan Dumanski, you have been an inspiration and a motiva-tor. Thank you for always believing in me and for constantly giving me the opportunities and the means to develop as a scientist. I’m grateful to have had the chance to do my PhD under your supervision. My co-supervisor, Lars Forsberg, thank you very much for all the produc-tive scientific discussions, I always appreciate all the conversations that we have had. My second co-supervisor, Tobias Sjöblom, thanks for the general support. My examiner, Niklas Dahl, thanks for your advices and for your supervision during the project that I did in your lab some years before the start of my PhD. Prof. Eva Tiensuu Janson, thanks for accepting me into your lab for a short pre-PhD period. My former supervisors during the projects before starting my PhD, Prof. Ulf Landegren and Associate Prof. Jonas Muhr, thank you for your supervi-sion and advices. My first supervisor, Associate Prof. Mohammad Reza Abbaszadegan, thanks for your supervision. Associate Prof. Lena Åslund and Associate Prof. Masood Kamali-Moghaddam, thanks for all your help. The administrative people at IGP, especially Christina Andersson, Ulla Steimer, and Gunilla Åberg for always being helpful. Teresita for your help when I started. Former and present members of the Dumanski and Diaz de Ståhl groups: Chiara, Hanna, Geeta, Johanna,

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Helena, Uwe, Cihan, Mehdi, Sai, Julia, Ravi, Reddy, Mana, Hanna, An-na and Szymon. The teaching team in the first year, Nikki, thanks for all your other help out-side the teaching world, Madhu and Victoria as well as the teaching crew in the second year, Anja, Lucy and Vasil. All collaborators, for your great contribution to my projects. All colleagues at the Rudbeck laboratory for creating a great and productive environment. My family for all the encouragements and for believing in me. Sist men inte minst, Julia. Du är min bästa vän. Jag uppskattar ditt tålamod. Kanske finns det avstånd nu, men snart kommer jag att vara nära dig. Stort tack för allt ditt stöd !

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Post-zygotic Genetic Variation in Health and Disease609581/FULLTEXT01.pdfPost-zygotic genetic variation, twins as a model in studies of post zygotic variation and the methods involved