Computational Methods for Detecting Large-Scale Chromosome Rearrangements in SNP Data

Large-scale chromosome rearrangements such as copy number variants (CNVs) and inversions encompass a considerable proportion of the genetic variation between human individuals. In a number of cases, they have been closely linked with various inheritable diseases. Single-nucleotide polymorphisms (SNPs) are another large part of the genetic variance between individuals. They are also typically abundant and their measuring is straightforward and cheap. This thesis presents computational means of using SNPs to detect the presence of inversions and deletions, a particular variety of CNVs. Technically, the inversion-detection algorithm detects the suppressed recombination rate between inverted and non-inverted haplotype populations whereas the deletion-detection algorithm uses the EM-algorithm to estimate the haplotype frequencies of a window with and without a deletion haplotype. As a contribution to population biology, a coalescent simulator for simulating inversion polymorphisms has been developed. Coalescent simulation is a backward-in-time method of modelling population ancestry. Technically, the simulator also models multiple crossovers by using the Counting model as the chiasma interference model. Finally, this thesis includes an experimental section. The aforementioned methods were tested on synthetic data to evaluate their power and specificity. They were also applied to the HapMap Phase II and Phase III data sets, yielding a number of candidates for previously unknown inversions, deletions and also correctly detecting known such rearrangements.

Molecular mechanisms of cancer predisposition in HNPCC/Lynch syndrome

Hereditary non-polyposis colorectal carcinoma (HNPCC; Lynch syndrome) is among the most common hereditary cancers in man and a model of cancers arising through deficient DNA mismatch repair (MMR). It is inherited in a dominant manner with predisposing germline mutations in the MMR genes, mainly MLH1, MSH2, MSH6 and PMS2. Both copies of the MMR gene need to be inactivated for cancer development. Since Lynch syndrome family members are born with one defective copy of one of the MMR genes in their germline, they only need to acquire a so called second hit to inactivate the MMR gene. Hence, they usually develop cancer at an early age. MMR gene inactivation leads to accumulation of mutations particularly in short repeat tracts, known as microsatellites, causing microsatellite instability (MSI). MSI is the hallmark of Lynch syndrome tumors, but is present in approximately 15% of sporadic tumors as well. There are several possible mechanisms of somatic inactivation (i.e. the second hit ) of MMR genes, for instance deletion of the wild-type copy, leading to loss of heterozygosity (LOH), methylation of promoter regions necessary for gene transcription, or mitotic recombination or gene conversion. In the Lynch syndrome tumors carrying germline mutations in the MMR gene, LOH was found to be the most frequent mechanism of somatic inactivation in the present study. We also studied MLH1/MSH2 deletion carriers and found that somatic mutations identical to the ones in the germline occurred frequently in colorectal cancers and were also present in extracolonic Lynch syndrome-associated tumors. Chromosome-specific marker analysis implied that gene conversion, rather than mitotic recombination or deletion of the respective gene locus accounted for wild-type inactivation. Lynch syndrome patients are predisposed to certain types of cancers, the most common ones being colorectal, endometrial and gastric cancer. Gastric cancer and uroepithelial tumors of bladder and ureter were observed to be true Lynch syndrome tumors with MMR deficiency as the driving force of tumorigenesis. Brain tumors and kidney carcinoma, on the other hand, were mostly MSS, implying the possibility of alternative routes of tumor development. These results present possible implications in clinical cancer surveillance. In about one-third of families suspected of Lynch syndrome, mutations in MMR genes are not found, and we therefore looked for alternative mechanisms of predisposition. According to our results, large genomic deletions, mainly in MSH2, and germline epimutations in MLH1, together explain a significant fraction of point mutation-negative families suspected of Lynch syndrome and are associated with characteristic clinical and family features. Our findings have important implications in the diagnosis and management of Lynch syndrome families.

Fumarate Hydratase and Succinate Dehydrogenase in Neoplasia

Germline mutations in fumarate hydratase (FH) cause hereditary leiomyomatosis and renal cell cancer (HLRCC). FH is a nuclear encoded enzyme which functions in the Krebs tricarboxylic acid cycle, and homozygous mutation in FH lead to severe developmental defects. Both uterine and cutaneous leiomyomas are components of the HLRCC phenotype. Most of these tumours show loss of the wild-type allele and, also, the mutations reduce FH enzyme activity, which indicate that FH is a tumour suppressor gene. The renal cell cancers associated with HLRCC are of rare papillary type 2 histology. Other genes involved in the Krebs cycle, which are also implicated in neoplasia are 3 of the 4 subunits encoding succinate dehydrogenase (SDH); mutations in SHDB, SDHC, and SDHD predispose to paraganglioma and phaeochromocytoma. Although uterine leiomyomas (or fibroids) are very common, the estimations of affected women ranging from 25% to 77%, not much is known about their genetic background. Cytogenetic studies have revealed that rearrangements involving chromosomes 6, 7, 12 and 14 are most commonly seen in fibroids. Deletions on the long arm of chromosome 7 have been reported to be involved in about 17 to 34 % of leiomyomas and the small commonly deleted region on 7q22 suggests that there might be an underlying tumour suppressor gene in that region. The purpose of this study was to investigate the genetic mechanisms behind the development of tumours associated with HLRCC, both renal cell cancer and uterine fibroids. Firstly, a database search at the Finnish cancer registry was conducted in order to identify new families with early-onset RCC and to test if the family history was compatible with HLRCC. Secondly, sporadic uterine fibroids were tested for deletions on 7q in order to define the minimal deleted 7q-region, followed by mutation analysis of the candidate genes. Thirdly, oligonucleotide chips were utilised to study the global gene expression profiles of uterine fibroids in order to test whether 7q-deletions and FH mutations significantly affected fibroid biology. In the screen for early-onset RCC, 214 families were identified. Subsequently, the pedigrees were constructed and clinical data obtained. One of the index cases (RCC at the age of 28) had a mother who had been diagnosed with a heart tumour, which in further investigation turned out to be a paraganglioma. This lead to an alternative hypothesis that SDH, instead of FH, could be involved. SDHA, SDHB, SDHC and SDHD were sequenced from these individuals; a germline SDHB R27X mutation was detected with loss of the wild-type allele in both tumours. These results suggest that germline mutations in the SDHB gene predispose to early-onset RCC establishing a novel form of hereditary RCC. This has immediate clinical implications in the surveillance of patients suffering from early-onset RCC and phaeochromocytoma/paraganglioma. For the studies on sporadic uterine fibroids, a set of 166 fibroids from 51 individuals were collected. The 7q LOH mapping defined a commonly deleted region of about 3.2 mega bases in 11 of the 166 tumours. The deletion was consistent with previously reported allelotyping studies of leiomyomas and it therefore suggested the presence of a tumour suppressor gene in the deleted region. Furthermore, the high-resolution aCGH-chip analysis refined the deleted region to only 2.79Mb. When combined with previous data, the commonly deleted region was only 2.3Mb. The mutation screening of the known genes within the commonly deleted region did not reveal pathogenic mutations, however. The expression microarray analysis revealed that FH-deficient fibroids, both sporadic and familial, had their distinct gene expression profile as they formed their own group in the unsupervised clustering. On the other hand, the presence or absence of 7q-deletions did not significantly alter the global gene expression pattern of fibroids, suggesting that these two groups do not have different biological backgrounds. Multiple differentially expressed genes were identified between FH wild-type and FH-mutant fibroids, and the most significant increase was seen in the expression of carbohydrate metabolism-related and hypoxia inducible factor (HIF) target genes.

The Role of Genes on Chromosome Locus 4q12 in Human Central Nervous System Tumors

Human central nervous system (CNS) tumors are a heterogeneous group of tumors occurring in brain, brainstem and spinal cord. Malignant gliomas (astrocytic and oligodendroglial tumors), which arise from the neuroepithelial cells are the most common CNS neoplasms in human. Malignant gliomas are highly aggressive and invasive tumors, and have a very poor prognosis. The development and progression of gliomas involve a stepwise accumulation of genetic alterations that generally affect either signal transduction pathways activated by receptor tyrosine kinases (RTKs), or cell cycle arrest pathways. Constitutive activation or deregulated signaling by RTKs is caused by gene amplification, overexpression or mutations. The aberrant RTK signaling results in turn in the activation of several downstream pathways, which ultimately lead to malignant transformation and tumor proliferation. Many genetic abnormalities implicated in nervous system tumors involve the genes located at the chromosomal region 4q12. This locus harbors the receptor tyrosine kinases KIT, PDGFRA and VEGFR2, and other genes (REST, LNX1) with neural function. Gene amplification and protein expression of KIT, PDGFRA, and VEGFR2 was studied using clinical tumor material. REST and LNX1, as well as NUMBL, the interaction partner of LNX1, were studied for their gene mutations and amplifications. In our studies, amplification of LNX1 was associated with KIT and PDGFRA amplification in glioblastomas, and coamplification of KIT, PDGFRA and VEGFR2 was detected in medulloblastomas and CNS primitive neuroectodermal tumors. PDGFRA amplification was also correlated with poor overall survival. Coamplification of KIT, PDGFRA and VEGFR2 was observed in a subset of human astrocytic and oligodendroglial tumors. We suggest that genes at 4q12 could be a part of a larger amplified region, which is deregulated in gliomas, and could be used as a prognostic marker of tumorigenic process. The signaling pathways activated due to gene amplifications, activating gene mutations, and overexpressed proteins may be useful as therapeutic targets for glioma treatment. This study also includes the characterization of KIT overexpressing astrocytes, analyzed by various in vitro functional assays. Our results show that overexpression of KIT in mouse astrocytes promotes cell proliferation and anchorage-independent growth, as well as phenotypic changes in the cells. Furthermore, the increased proliferation is partly inhibited by imatinib, a small molecule inhibitor of KIT. These results suggest that KIT may play a role in astrocyte growth regulation, and might have an oncogenic role in brain tumorigenesis. Elucidation of the altered signaling pathways due to specific gene amplifications, activating gene mutations, and overexpressed proteins may be useful as therapeutic targets for glioma treatment.

Microarrays in Molecular Profiling of Ewing Sarcoma Family of Tumors and CDKN2A Aberrations

Background: The Ewing sarcoma family of tumors (ESFT) are rare but highly malignant neoplasms that occur mainly in bone or but also in soft tissue. ESFT affects patients typically in their second decade of life, whereby children and adolescents bear the heaviest incidence burden. Despite recent advances in the clinical management of ESFT patients, their prognosis and survival are still disappointingly poor, especially in cases with metastasis. No targeted therapy for ESFT patients is currently available. Moreover, based merely on current clinical and biological characteristics, accurate classification of ESFT patients often fails at the time of diagnosis. Therefore, there is a constant need for novel molecular biomarkers to be applied in tandem with conventional parameters to further intensify ESFT risk-stratification and treatment selection, and ultimately to develop novel targeted therapies. In this context, a greater understanding of the genetics and immune characteristics of ESFT is needed. Aims: This study sought to open novel insights into gene copy number changes and gene expression in ESFT and, further, to enlighten the role of inflammation in ESFT. For this purpose, microarrays were used to provide gene-level information on a genomewide scale. In addition, this study focused on screening of 9p21.3 deletion sizes and frequencies in ESFT and, in another pediatric cancer, acute lymphocytic leukemia (ALL), in order to define more exact criteria for highrisk patient selection and to provide data for developing a more reliable diagnostic method to detect CDKN2A deletions. Results: In study I, 20 novel ESFT-associated suppressor genes and oncogenes were pinpointed using combined array CGH and expression analysis. In addition, interesting chromosomal rearrangements were identified: (1) Duplication of derivative chromosome der(22)(11;22) was detected in three ESFT patients. This duplication included the EWSR1-FLI1 fusion gene leading to increase in its copy number; (2) Cryptic amplifications on chromosomes 20 and 22 were detected, suggesting a novel translocation between chromosomes 20 and 22, which most probably produces a fusion between EWSR1 and NFATC2. In study II, bioinformatic analysis of ESFT expression profiles showed that inflammatory gene activation is detectable in ESFT patient samples and that the activation is characterized by macrophage gene expression. Most interestingly, ESFT patient samples were shown to express certain inflammatory genes that were prognostically significant. High local expression of C5 and JAK1 at the tumor site was shown to associate with favorable clinical outcome, whereas high local expression of IL8 was shown to be detrimental. Studies III and IV showed that the smallest overlapping region of deletion in 9p21.3 includes CDKN2A in all cases and that the length of this region is 12.2 kb in both Ewing sarcoma and ALL. Furthermore, our results showed that the most widely used commercial CDKN2A FISH probe creates false negative results in the narrowest microdeletion cases (<190 kb). Therefore, more accurate methods should be developed for the detection of deletions in the CDKN2A locus. Conclusions: This study provides novel insights into the genetic changes involved in the biology of ESFT, in the interaction between ESFT cells and immune system, and in the inactivation of CDKN2A. Novel ESFT biomarker genes identified in this study serve as a useful resource for future studies and in developing novel therapeutic strategies to improve the survival of patients with ESFT.

Molecular genetics of tooth agenesis

Congenital missing of teeth, tooth agenesis or hypodontia, is one of the most common developmental anomalies in man. The common forms in which one or a few teeth are absent, may cause occlusal or cosmetic harm, while severe forms which are relatively rare always require clinical attention to support and maintain the dental function. Observation of tooth agenesis is also important for diagnosis of malformation syndromes. Some external factors may cause developmental defects and agenesis in dentition. However, the role of inheritance in the etiology of tooth agenesis is well established by twin and family studies. Studies on familial tooth agenesis as well as mouse null mutants have also identified several genetic factors. However, these explain syndromic or rare dominant forms of tooth agenesis, whereas the genes and defects responsible for the majority of cases of tooth agenesis, especially the common and less severe forms, are largely unknown. In this study it was shown, that a dominant nonsense mutation in PAX9 was responsible for severe tooth agenesis (oligodontia) in a Finnish family. In a study of tooth agenesis associated with Wolf-Hirschhorn syndrome, it was shown that severe tooth agenesis was present if the causative deletion in 4p spanned the MSX1 locus. It was concluded that severe tooth agenesis was caused by haploinsufficiency of these transcription factors. A summary of the phenotypes associated with known defects in MSX1 and PAX9 showed that, despite similarities, they were significantly different, suggesting that the genes, in addition to known interactions, also have independent roles during the development of human dentition. The original aim of this work was to identify gene defects that underlie the common incisor and premolar hypodontia. After excluding several candidate genes, a genome-wide search was conducted in seven Finnish families in which this phenotype was inherited in an autosomal dominant manner. A promising locus for second premolar agenesis was identified in chromosome 18 in one family and this finding was supported by results from other families. The results also implied the existence of other loci both for second premolar agenesis and for incisor agenesis. On the other hand the results did not lend support for comprehensive involvement of the most obvious candidate genes in the etiology of incisor and premolar hypodontia. Rather, they suggest remarkable genetic heterogeneity of tooth agenesis. The available evidence suggests that quantitative defects during tooth development predispose to a failure to overcome a developmental threshold and to agenesis. The results of the study increase the understanding of the etiology and heredity of tooth agenesis. Further studies may lead to identification of novel genes that affect the development of teeth.

GPRA and the asthma locus on chromosome 7p14-p15

The basis of this work was the identification of a genomic region on chromosome 7p14-p15 that strongly associated with asthma and high serum total immunoglobulin E in a Finnish founder population from Kainuu. Using a hierarchical genotyping approach the linkage region was narrowed down until an evolutionary collectively inherited 133-kb haplotype block was discovered. The results were confirmed in two independent data sets: Asthma families from Quebec and allergy families from North-Karelia. In all the three cohorts studied, single nucleotide polymorphisms tagging seven common gene variants (haplotypes) were identified. Over half of the asthma patients carried three evolutionary closely related susceptibility haplotypes as opposed to approximately one third of the healthy controls. The risk effects of the gene variants varied from 1.4 to 2.5. In the disease-associated region, there was one protein-coding gene named GPRA (G Protein-coupled Receptor for Asthma susceptibility also known as NPSR1) which displayed extensive alternative splicing. Only the two isoforms with distinct intracellular tail sequences, GPRA-A and -B, encoded a full-length G protein-coupled receptor with seven transmembrane regions. Using various techniques, we showed that GPRA is expressed in multiple mucosal surfaces including epithelial cells throughout the respiratory tract. GPRA-A has additional expression in respiratory smooth muscle cells. However, in bronchial biopsies with unknown haplotypes, GPRA-B was upregulated in airways of all patient samples in contrast to the lack of expression in controls. Further support for GPRA as a common mediator of inflammation was obtained from a mouse model of ovalbumin-induced inflammation, where metacholine-induced airway hyperresponsiveness correlated with elevated GPRA mRNA levels in the lung and increased GPRA immunostaining in pulmonary macrophages. A novel GPRA agonist, Neuropeptide S (NPS), stimulated phagocytosis of Esterichia coli bacteria in a mouse macrophage cell line indicating a role for GPRA in the removal of inhaled allergens. The suggested GPRA functions prompted us to study, whether GPRA haplotypes associate with respiratory distress syndrome (RDS) and bronchopulmonary dysplasia (BPD) in infants sharing clinical symptoms with asthma. According to the results, near-term RDS and asthma may also share the same susceptibility and protective GPRA haplotypes. As in asthma, GPRA-B isoform expression was induced in bronchial smooth muscle cells in RDS and BPD suggesting a role for GPRA in bronchial hyperresponsiveness. In conclusion, the results of the present study suggest that the dysregulation of the GPRA/NPS pathway may not only be limited to the individuals carrying the risk variants of the gene but is also involved in the regulation of immune functions of asthma.

Acute Lymphoblastic Leukemia in Adolescents and Young Adults in Finland

In recent reports, adolescents and young adults (AYA) with acute lymphoblastic leukemia (ALL) have had a better outcome with pediatric treatment than with adult protocols. ALL can be classified into biologic subgroups according to immunophenotype and cytogenetics, with different clinical characteristics and outcome. The proportions of the subgroups are different in children and adults. ALL subtypes in AYA patients are less well characterized. In this study, the treatment and outcome of ALL in AYA patients aged 10-25 years in Finland on pediatric and adult protocols was retrospectively analyzed. In total, 245 patients were included. The proportions of biologic subgroups in different age groups were determined. Patients with initially normal or failed karyotype were examined with oligonucleotide microarray-based comparative genomic hybridization (aCGH). Also deletions and instability of chromosome 9p were screened in ALL patients. In addition, patients with other hematologic malignancies were screened for 9p instability. aCGH data were also used to determine a gene set that classifies AYA patients at diagnosis according to their risk of relapse. Receiver operating characteristic analysis was used to assess the value of the set of genes as prognostic classifiers. The 5-year event-free survival of AYA patients treated with pediatric or adult protocols was 67% and 60% (p=0.30), respectively. White blood cell count larger than 100x109/l was associated with poor prognosis. Patients treated with pediatric protocols and assigned to an intermediate-risk group fared significantly better than those of the pediatric high-risk or adult treatment groups. Deletions of 9p were detected in 46% of AYA ALL patients. The chromosomal region 9p21.3 was always affected, and the CDKN2A gene was always deleted. In about 15% of AYA patients, the 9p21.3 deletion was smaller than 200 kb in size, and therefore, probably undetectable with conventional methods. Deletion of 9p was the most common aberration of AYA ALL patients with initially normal karyotype. Instability of 9p, defined as multiple separate areas of copy number loss or homozygous loss within a larger heterozygous area in 9p, was detected in 19% (n=27) of ALL patients. This abnormality was restricted to ALL; none of the patients with other hematologic malignancies had the aberration. The prognostic model identification procedure resulted in a model of four genes: BAK1, CDKN2B, GSTM1, and MT1F. The copy number profile combinations of these genes differentiated between AYA ALL patients at diagnosis depending on their risk of relapse. Deletions of CDKN2B and BAK1 in combination with amplification of GSTM1 and MT1F were associated with a higher probability of relapse. Unlike all previous studies, we found that the outcome of AYA patients with ALL treated using pediatric or adult therapeutic protocols was comparable. The success of adult ALL therapy emphasizes the benefit of referral of patients to academic centers and adherence to research protocols. 9p deletions and instability are common features of ALL and may act together with oncogene-activating translocations in leukemogenesis. New and more sensitive methods of molecular cytogenetics can reveal previously cryptic genetic aberrations with an important role in leukemic development and prognosis and that may be potential targets of therapy. aCGH also provides a viable approach for model design aiming at evaluation of risk of relapse in ALL.

Molecular diagnosis of developmental disorders

ABSTRACT Idiopathic developmental disorders (DDs) affect ~1% of the population worldwide. This being a considerable amount, efforts are being made to elucidate the disease mechanisms. One or several genetic factors cause 30-40% of DDs, and only 10% are caused by environmental factors. The remaining 50% of DD patients go undiagnosed, mostly due to a lack of diagnostic techniques. The cause in most undiagnosed cases is though to be a genetic factor or a combination of genetic and environmental factors. Despite the surge of new technologies entering the market, their implementation into diagnostic laboratories is hampered by costs, lack of information about the expected diagnostic yield, and the wide range of selection. This study evaluates new microarray methods in diagnosing idiopathic DDs, providing information about their added diagnostic value. Study I analysed 150 patients by array comparative genomic hybridization (array CGH, 44K and 244K), with a subsequent 18% diagnostic yield. These results are supported by other studies, indicating an enourmous added diagnostic value of array CGH, compared with conventional cytogenetic analysis. Nevertheless, 80% of the patients remained undiagnosed in Study I. In an effort to diagnose more patients, in Study IV the resolution was increased from 8.9 Kb of the 244K CGH array to 0.7 Kb, by using a single-nucleotide polymorphism (SNP) array. However, no additional pathogenic changes were detected in the 35 patients assessed, and thus, for diagnostic purposes, an array platform with ca 9 Kb resolution appears adequate. The recent vast increase in reports of detected aberrations and associated phenotypes has enabled characterization of several new syndromes first based on a common aberration and thereafter by delineation of common clinical characteristics. In Study II, a familial deletion at 9q22.2q22.32 with variable penetrance was described. Despite several reports of aberrations in the adjacent area at 9q associated with Gorlin syndrome, the patients in this family had a unique phenotype and did not present with the syndrome. In Study III, a familial duplication of chromosome 6p22.2 was described. The duplication caused increased expression of an important enzyme of the γ-aminobutyric acid (GABA) degradation pathway, causing oxidative stress of the brain, and thus, very likely, the mild mental retardation of these patients. These two case studies attempted to pinpoint candidate genes and to resolve the pathogenic mechanism causing the clinical characteristics of the patients. Presenting rare genetic and clinical findings to the international science and medical community enables interpretation of similar findings in other patients. The added value of molecular karyotyping in patients with idiopathic DD is evident. As a first line of testing, arrays with a median resolution of at least 9 Kb should be considered and further characterization of detected aberrations undertaken when possible. Diagnostic whole-exome sequencing may be the best option for patients who remain undiagnosed after high-resolution array analysis.