A large brain is one of the most distinguishing features of humans compared to other members of the animal kingdom. During mammalian evolution there has been a disproportionate enlargement of the brain relative to body size and this expansion has been particularly prominent during the past 3 million years of human lineage. This must be the consequence of adaptive genetic alterations during mammalian evolution, but the genes and molecular processes altered are essentially unknown. One approach for identifying candidate genes for brain size regulation is through characterisation of Mendelian disorders of brain development. In particular, primary microcephaly has received considerable interest as a model disease for studying brain size regulators because patients present with a profoundly reduced brain size but have no other malformations. Genetic studies have identified mutations in seven genes that can cause primary microcephaly. All the primary microcephaly proteins localise to the centrosome at some stage during the cell cycle and have roles in a diverse range of functions including centrosome maturation, centriole formation and microtubule organisation at the spindle pole. The precise mechanism leading to primary microcephaly is not known but a prevalent hypothesis is that centrosome dysfunction disrupts mitosis of neural progenitor cells. Despite there being strong evidence in support of this hypothesis for most primary microcephaly genes, MCPH1 (the first primary microcephaly gene to be identified) always appeared to be functionally distinct from other primary microcephaly proteins. Most work on MCPH1 has focussed on its role in the DNA damage response and cell cycle timing rather than on its mitotic role. As a result, the aim of this thesis is to perform a detailed analysis of MCPH1 function during mitosis. In this thesis, three isoforms of MCPH1 were characterised and their localisation, expression and stability examined. It was established that MCPH1 is highly regulated during mitosis. MCPH1 transcript and protein levels vary significantly throughout the cell cycle and MCPH1 protein is targeted for degradation late in mitosis. In addition, MCPH1 is hyperphosphorylated during mitosis (in prometaphase-arrested cells) suggesting that phosphorylation could potentially regulate MCPH1 mitotic function. Twelve mitotic phosphorylation sites were identified by phosphopeptide mapping, many of which were CDK1 and PLK1 consensus sites. Both PLK1 and CDK1 also contribute to MCPH1 phosphorylation in vivo. Although MCPH1 non-phosphorylatable mutants localise normally during mitosis, binding to interaction partners may be affected which may have functional consequences. During mitosis MCPH1 localises to the centrosomes and kinetochores. Consistent with this localisation, RNAi-mediated knockdown of MCPH1 leads to metaphase arrest with multipolar spindles, major defects in chromosome alignment and loss of chromatid cohesion. In addition, MCPH1 deficient mouse embryonic fibroblast cells also demonstrate similar chromosome alignment defects, strengthening this finding in an independent system. Live-imaging of MCPH1 depleted cells demonstrate that a normal bipolar spindle and metaphase plate are initially formed, but subsequently chromosomes and chromatids drop off the metaphase plate and eventually the spindle collapses. This suggests that the primary function of MCPH1 is to allow timely progression through metaphase, possibly by mediating kinetochore-microtubule attachments to satisfy the spindle activated checkpoint. Therefore my work describes several roles for MCPH1 in mitosis (centrosome stability, chromosome alignment and metaphase progression) suggesting that its role in mitosis could result in primary microcephaly in a number of different ways.

Abstract Breast cancer is the most common malignancy in women and it is strongly influenced by hereditary risk factors. So far, most of the breast cancer-associated genes, including BRCA1/2, have been identified among those that encode proteins involved in DNA damage response (DDR) pathways. However, known genetic risk factors explain less than half of the familial risk of breast cancer. Identification of novel genes and mutations that predispose to breast cancer is important for the understanding of the mechanisms that contribute to the disease development and also for the identification of those individuals who are at high risk. The first aim of this study was to resolve the complementation groups of Finnish patients with Fanconi anemia (FA), which is a rare genetic disease caused by defects in a specific DDR pathway, and to study the role of the causative gene mutations in breast cancer predisposition. The second aim of this study was to identify novel breast cancer susceptibility genes and alleles by targeted next-generation sequencing (NGS) of multiple (~800) DDR related genes. In both approaches, the identified gene mutations were subjected to case-control association analysis utilizing DNA samples of over 1,000 breast cancer cases and 1,000 healthy controls. Investigation of the Finnish FA patients revealed six different disease-causing mutations in three different genes (FANCA, FANCG and FANCI). All of the studied mutations were recurrent in the Finnish population but did not associate with breast cancer. Targeted NGS identified three novel potential breast cancer susceptibility genes. A significant enrichment of TEX15 c.7253dupT and FANCD2 c.2715+1G>A mutations was observed among the hereditary breast cancer cases (P = 0.018 and P = 0.036, respectively). The strongest evidence was found for a Finnish founder mutation in MCPH1 (c.904_916del), which associated with breast cancer susceptibility both in familial (P = 0.003, OR 8.3) and unselected (P = 0.016, OR 3.3) patient cohorts. The tumor suppressive function of MCPH1 was indicated by the loss of the wild-type allele of MCPH1 in 40% of the studied carrier tumors. Furthermore, carriers exhibited a significant increase in genomic instability measured by spontaneous chromosomal rearrangements in peripheral blood lymphocytes. / Tiivistelmä Rintasyöpä on naisten yleisin syöpä. Sairastumisriskiin vaikuttavat voimakkaasti perinnölliset alttiustekijät, ja suurin osa tähän asti tunnistetuista rintasyöpäalttiusgeeneistä, kuten BRCA1/2, koodaa DNA-vauriovasteessa (DDR) toimivia proteiineja. Tunnistetut tekijät selittävät yhä kuitenkin vain alle puolet rintasyövän perinnöllisestä alttiudesta. Uusien alttiusgeenien tunnistaminen on tärkeää rintasyövän patomekanismien ymmärtämiseksi sekä korkean rintasyöpäriskin omaavien henkilöiden tunnistamiseksi. Tämän tutkimuksen tarkoituksena oli määrittää viallisesta DDR-signaalinsiirtoreitistä johtuvan Fanconin anemian (FA) komplementaatioryhmät suomalaisilta FA-potilailta sekä tutkia sairauden taustalla olevien geenimutaatioden yhteyttä rintasyöpäriskiin. Uusia alttiusgeenejä etsittiin myös kohdennetulla uuden sukupolven sekvensointimenetelmällä, jonka avulla tutkittiin yhtäaikaisesti n. 800 DDR-geeniä. Molemmilla lähestymistavoilla tunnistettujen geenimuutosten yhteyttä rintasyöpään selvitettiin tapaus-verrokkitutkimuksen avulla, jossa tutkittiin DNA-näytteitä yli tuhannelta rintasyöpäpotilaalta sekä yli tuhannelta terveeltä henkilöltä. Suomalaisten FA-potilaiden geenimuutoksia selvittävässä tutkimuksessa tunnistettiin yhteensä kuusi mutaatiota kolmessa eri geenissä (FANCA, FANCG ja FANCI). Kaikki tutkimuksessa tunnistetut mutaatiot olivat toistuvia suomalaisessa väestössä, mutta merkitsevää assosiaatiota näiden mutaatioiden ja rintasyöpäalttiuden välillä ei havaittu. DDR-geenien sekvensoinnin avulla tunnistettiin kolme uutta mahdollista rintasyöpäalttiusgeeniä. Tutkimuksessa havaittiin TEX15 c.7253dupT ja FANCD2 c.2715+1G>A mutaatioiden rikastuminen perinnöllisessä rintasyöpäaineistossa (P = 0.018 ja P = 0.036). Merkittävin yhteys rintasyöpäalttiuden kanssa todettiin MCPH1-geenin perustajamutaatiolle (c.904_916del). Tämä mutaatio assosioitui rintasyöpäalttiuden kanssa sekä perinnöllisessä (P = 0.003, OR 8.3) että valikoimattomassa potilasaineistossa (P = 0.016, OR 3.3). Useissa mutaatiokantajien tuumoreissa (40 %) normaali MCPH1 vastinalleeli oli hävinnyt, mikä viittaisi siihen, että MCPH1 toimii tuumorisuppressorina. Mutaatiokantajilla todettiin myös kohonnut määrä kromosomaalisia muutoksia veren periferaalisissa lymfosyyteissä, mahdollisesti kohonneeseen genomiseen epävakauteen liittyen.

Autosomal recessive primary microcephaly (MCPH) is a congenital neurodevelopmental disorder characterised by a reduced occipital-frontal head circumference (OFC) of less than -3 SDs below the population mean for age and sex. It is a genetically heterogeneous disorder caused by mutations in one of the following 10 MCPH genes: MCPH1 (microcephalin 1), WDR62 (WD repeat domain 62), CDK5RAP2 (cyclin-dependent kinase 5 regulatory associated protein 2), CASC5 (cancer susceptibility candidate 5), CEP152 (centrosomal protein 152 kDa), ASPM (asp [abnormal spindle] homolog, microcephaly associated [Drosophila]), CENPJ (centromeric protein J), STIL (SCL/TAL1-interrupting locus), CEP135 (centrosomal protein 135 kDa) and CEP63 (centrosomal protein 135 kDa). The MCPH1 (microcephalin 1) gene is located on chromosome 8p23.1. Microsatellite analysis has previously shown LOH at the markers D8S518 and D8S277 flanking the MCPH1 locus in 1/21 oral tumors. Furthermore, LOH at the markers D8S1742 and D8S277 flanking the MCPH1 locus has also been observed in 2/32 hepatocellular carcinomas. MCPH1 has been found to be mutated in breast and endometrial cancers. Additionally, it was found to be downregulated at the transcript level in 19/30 ovarian cancer tissues and the protein level in 93/319 breast cancer tissues. Decreased MCPH1 protein levels are associated with triple negative breast cancers and a lower transcript level of MCPH1 correlates with lesser time for metastasis to occur in breast cancer patients. Interestingly, MCPH1 knockout mice in a null TP53 background show susceptibility to cancer.So far, studies have indicated that MCPH1 is a DNA repair protein. MCPH1 is required for the formation of DNA repair foci, chromatin relaxation, HR and NHEJ. It regulates G1/S and G2/M cell cycle checkpoints. Also, depletion of MCPH1 leads to genomic instability and centrosome amplification. Hence, the defect in the function of MCPH1 can lead to plethora of anomalies including cancer. Based on these observations, we hypothesized that MCPH1 may also function as a tumor suppressor (TS) gene, in addition to its role in the brain development. The purpose of this study was to test if MCPH1 also functions as a TS gene using different approaches in OSCC (oral squamous cell carcinoma). OSCC is the sixth most common type of cancer. It includes the cancer of the lips, anterior 2/3rd of the tongue, buccal mucosa, floor of the mouth, retromolar trigone and gingiva. Despite the advances in the treatment of oral cancer, the five-yr survival rate has not increased. Hence, the effective treatment of OSCC requires the identification of molecular targets to design appropriate therapeutic strategies. LOH, mutations and promoter methylation in tumors are the hallmarks of TS genes. In order to ascertain the TS roles of MCPH1, we carried out LOH analysis in 81 matched blood/normal and tumor oral tissues using D8S1819, D8S277 and D8S1798 markers flanking the MCPH1 locus. The results showed LOH at one or more markers in 14/71 (19.72%) informative samples across the tumor stages from T1 to T4. The entire coding region and the exon-intron junctions of the MCPH1 gene were sequenced for mutations in 15 OSCC samples and 5 cancer cell lines (viz., A549, HeLa, KB, SCC084 and SCC131). In total, three mutations namely c.1561G>T(p.Glu521X), c.321delA(p.Lys107fsX39) and c.1402delA(p.Thr468fsX32) were identified. The expression of MCPH1 was analysed at both the transcript and protein levels by real-time quantitative RT-PCR and immunohistochemistry, respectively, in OSCC samples. MCPH1 was downregulated in 51.22% (21/41) of OSCC samples at the transcript level. The MCPH1 protein was downregulated in 76% (19/25) of the OSCC samples. In order to elucidate if the MCPH1 promoter was methylated in OSCC tissues, we retrieved the MCPH1 promoter from the database TRED (Transcriptional Regulatory Element Database). The promoter was analysed for the presence of CpG islands using the CpG Plot/CpG Report program. Two CpG islands (CpGI and CpGII) were identified within the MCPH1 promoter. Both the CpG islands were analysed for methylation in 40 OSCC samples by COBRA (Combined Bisulfite Restriction Analysis). CpGI showed no methylation in 40 OSCC samples. However, CpGII showed methylation in 4/40 (10%) OSCC samples and the methylation was absent in their corresponding normal oral tissues. To analyse the methylation of the MCPH1 promoter in cancer cell lines, HeLa, KB, SCC084 and SCC131 cells were treated with 5’-2-deoxy azacytidine (AZA), a methyltrasferase inhibitor. HeLa and KB cells did not show any change in the MCPH1 transcript level after the AZA treatment. However, SCC084 and SCC131 cells showed upregulation of MCPH1 after the treatment, suggesting methylation of the MCPH1 promoter. To validate these observations, we examined the methylation status of both the CpG islands in these cell lines. We found methylation of CpGII only in SCC084 cells. HeLa, KB and SCC131 cells showed no methylation of CpGI and CpGII. The results obtained by COBRA in these cell lines were further confirmed by bisulfite sequencing of CpGI and CpGII islands. Further, the upregulation of MCPH1 after azacytidine treatment in SCC131 cells can be attributed to a promoter independent mechanism or due to methylation of the CpG sites not examined by us. To elucidate the biological effects of MCPH1 in a cancer cell line, we generated stable clones overexpressing MCPH1 in KB cells. The results showed that MCPH1 overexpression decreased cellular proliferation, cell invasion, anchorage-independent growth in soft-agar and tumor growth in nude mice. Further, MCPH1 overexpression lead to apoptosis. A low frequency of LOH, mutations and promoter methylation suggested that they might not be the major mechanisms of downregulation of MCPH1 in OSCC. We then speculated that MCPH1 could be regulated by miRNAs. We therefore used five miRNA target prediction softwares to identify miRNAs targeting MCPH1. The programs identified two binding sites for miR-27a within the 5.4 kb region of the 3’-UTR of MCPH1. The luciferase assay showed that both the seed regions of MCPH1 were binding to miR-27a. In addition, transient transfection of the premiR-27a construct in KB cells decreased the protein level of MCPH1. Additionally, in a small panel of 10 OSCC samples, there was a negative correlation between the levels of miR-27a and MCPH1. To the best of our knowledge, this is the first report showing any miRNA regulating the MCPH1 gene. It is important to note that tumor suppressors can serve as potential biomarkers with prognostic value. Hence, we analysed the correlation of the expression levels of MCPH1 with clinico-pathological parameters such as TNM, gender, age and site of the cancer by Fischer’s exact test. No statistical correlation was observed between the transcript or protein levels with any of the clinico-pathological parameters. In summary, the results of the present study have suggested that the primary microcephaly gene MCPH1 shows several hallmarks of TS genes and functions as a tumor suppressor in OSCC, in addition to its role in brain development. We have for the first time shown that miR-27a targets MCPH1 and regulates its level. It is interesting to note that none of the other 10 MCPH genes have been shown to be regulated by any miRNA yet. Our study will be useful in designing novel therapeutic methods for the treatment of OSCC either by overexpression of MCPH1 or reducing the level of miR-27a by an antagomir.