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Faktör VIII geninde en sıklıkla görülen mutasyonlardan ikisi, intron 22 deki inversiyon mutasyonu ile CpG dinükleotidlerindeki C- »T değişimleridir. Her iki tip mutasyon bu tez kapsamında çalışılmıştır. İnversiyon mutasyonunu saptamak için hemofili A hastalyğı görülen Türk ailelerinin % 25 'ine kesin tanı verme olanağı sağlayan bir Southern-blot yöntemi uygulanmıştır. Buna ek olarak, iki genetik markör, intron 22 'deki mikrosatelit tekrarı ve Xba I kesim bölgesi incelenmiştir. Her iki markör için de heterozigot oranı yaklaşık %50 olarak bulunmuştur, intron 13 ve intron 22'deki mikrosatelit tekrarları, Hind III RFLP ve St 14 VNTR markörleri ile birlikte kullanıldığında, Türk hemofili ailelerinin yaklaşık %93'üne bağlantı analizi ile tanı verilebileceği gösterilmiştir. Faktör VIII geninde rastlanan nokta mutasyonlarının %45'i CpG dinükleotitlerinde meydana gelmektedir. Şimdiye kadar yapılan bazı çalışmalarda CpG dinükleotidlerindeki C- »T mutasyonlannın daha çok paternel kökenli olduğuna işaret edilmekte ve bu verinin dişi üreme hücrelerinde görülen hipometilasyonun doğrudan bir sonucu olduğu düşünülmektedir. Ancak, Şimdiye kadar insan üreme hücrelerinde, hastalıklara yol açan kodonlarda, metilasyon derecesinde bir farklılık olduğunu gösteren bir kanıt yoktur. Bu çalışmada, Faktör VIII ve FGFR 3 genlerinin seçilmiş bölgelerinde, dişi ve erkek üreme hücrelerindeki metilasyon durumunu saptamak için yeni bir araştırma başlatılmıştır. Bu çalışma kümeleşmemiş CpG bölgelerinde metilasyonun, spermatositler, olgun yumurta ve polar yapılarda eşit düzeyde olduğunu göstermiştir. Alınan sonuçlar, sitozin metilasyonu ile CpG dinükleotidlerindeki hipermutabilite arasındaki ilişkiyi desteklemekte ve erkek üreme hücrelerindeki yüksek mutasyon oranının sadece cinsiyete bağlı metilasyon farklılığı ile açıklanamıyacağını göstermektedir. Aynca bu sonuçlar, sperm oluşumunda hücre bölünme sayısının çok fazla olmasının, erkek üreme hücrelerindeki yüksek mutasyon oranından sorumlu olan en önemli etken olduğuna işaret etmekte ve yüksek bölünme hızının, metilasyona uğramış CpG bölgelerinde C- »T dönüşümlerini nasıl etkilediği tartışmasını başlatmaktadır. The factor VIII gene includes two major mutation hotspots, namely the intron 22 inversion and transitions at CpG dinucleotides, both of which have been examined in the context of this thesis. A non-radioactive Southern blot approach was implemented for the detection of the invers:~n, that allowed an accurate diagnosis for about V* of the Turkish hemophilia A families. In addition to the studies on the inversion mutation, two additional genetic markers, located in intron 22, were investigated in the Turkish population. The heterozygosity of both, the microsatellite repeat and the Xba I RFLP, was about 50 percent. It is concluded that the combined use of the two microsatellite repeats in intron 13 and intron 22 together with Hind III RFLP and St 14 VNTR markers enables tracking of the defective allele and therefore, provides genetic counseling by linkage analysis in about 93 percent of Turkish hemophilia A families. About 45 percent of all point mutations in the factor VIII gene occur at CpG sites. A number of previous studies had pointed to a bias in paternal origin for transition mutation at CpG dinucleotides. This was believed to be a direct reflection of the hypomethylation in the female germ cells. However, to date there was no direct proof for a differential level of methylation in the germ cells, at human disease causing codons. In this study, an original investigation was undertaken to determine the methylation status of mature male and female germ cells at selected sites in the human factor VIII and the FGFR3 genes. An equally high level of methylation, at non-cluster CpG sites, was observed in mature eggs, spermatocytes and polar bodies. This result, while confirming the link between the hyper-mutability of CpG dinucleotides and cytosine methylation, directly shows that the higher mutation rate in the male germ line is apparently not a simple reflection of sex specific methylation differences. These points to the greater number of cell divisions during spermatogenesis, as the major factor that accounts for the higher mutability of male germ cells and opens the discussion on how a high replication rate induces higher C to T transitions at methylated CpG sites. 126

Biomaterials to be used as implants , in the human body, mustbe safe. These materials must never induce any biological rejectionor disorganized growth in the tissues in which they are immersed .In. this study the safety ( the biocompatibility ) of Boride andNitride coated 316 L surgical stainless steels are tested . For thistwo in vivo tests have been performed , a systemic toxicity testusing mice) , and a subcutaneous implantation test (using rats .)Histological observation of tissue around the coated samplesshowed a higher degree of response than the uncoated 316 L surgicalstainless steels . This higher degree of response was attributed tothe presence of foreign bodies around the coated implants .Based on these primary testings performed , it has been foundthat the coated implants were of a lesser degree of biocompatibilitythan the uncoated 316 L surgical stainless steel . 144

The angiotensin-converting enzyme (ACE) plays a central role in the renin-angiotensin system, which is involved in the regulation of blood pressure. Alterations in ACE expression or activity are associated with various pathological phenotypes, particularly cardiovascular diseases. In human endothelial cells, ACE was shown to be negatively regulated by tumor necrosis factor (TNF) α. To examine, whether or not, epigenetic factors were involved in ACE expression regulation, methylated DNA immunoprecipitation and RNA interference experiments directed against regulators of DNA methylation homeostasis i.e., DNA methyltransferases (DNMTs) and ten-eleven translocation methylcytosine dioxygenases (TETs), were performed. TNFα stimulation enhanced DNA methylation in two distinct regions within the ACE promoter via a mechanism linked to DNMT3a and DNMT3b, but not to DNMT1. At the same time, TET1 protein expression was downregulated. In addition, DNA methylation decreased the binding affinity of the transcription factor MYC associated factor X to the ACE promoter. In conclusion, DNA methylation determines the TNFα-dependent regulation of ACE gene transcription and thus protein expression in human endothelial cells.

DNA methylation is essential in mammalian development. We have hypothesized that methylation differences induced by trisomy 21 (T21) contribute to the phenotypic characteristics and heterogeneity in Down syndrome (DS). In order to determine the methylation differences in T21 without interference of the interindividual genomic variation, we have used fetal skin fibroblasts from monozygotic (MZ) twins discordant for T21. We also used skin fibroblasts from MZ twins concordant for T21, normal MZ twins without T21, and unrelated normal and T21 individuals. Reduced Representation Bisulfite Sequencing (RRBS) revealed 35 differentially methylated promoter regions (DMRs) (Absolute methylation differences = 25%, FDR < 0.001) in MZ twins discordant for T21 that have also been observed in comparison between unrelated normal and T21 individuals. The identified DMRs are enriched for genes involved in embryonic organ morphogenesis (FDR = 1.60 e -03) and include genes of the HOXB and HOXD clusters. These DMRs are maintained in iPS cells generated from this twin pair and are correlated with the gene expression changes. We have also observed an increase in DNA methylation level in the T21 methylome compared to the normal euploid methylome. This observation is concordant with the up regulation of DNA methyltransferase enzymes (DNMT3B and DNMT3L) and down regulation of DNA demethylation enzymes (TET2 and TET3) observed in the iPSC of the T21 versus normal twin. Altogether, the results of this study highlight the epigenetic effects of the extra chromosome 21 in T21 on loci outside of this chromosome that are relevant to DS associated phenotypes. Published version

Background Pseudohypoparathyroidism (PHP) indicates a group of heterogeneous disorders whose common feature is represented by impaired signaling of hormones that activate Gsalpha, encoded by the imprinted GNAS gene. PHP-Ib patients have isolated Parathormone (PTH) resistance and GNAS epigenetic defects while PHP-Ia cases present with hormone resistance and characteristic features jointly termed as Albright's Hereditary Osteodystrophy (AHO) due to maternally inherited GNAS mutations or similar epigenetic defects as found for PHP-Ib. Pseudopseudohypoparathyroidism (PPHP) patients with an AHO phenotype and no hormone resistance and progressive osseous heteroplasia (POH) cases have inactivating paternally inherited GNAS mutations. Methodology/Principal Findings We here describe 17 subjects with an AHO-like phenotype that could be compatible with having PPHP but none of them carried Gsalpha mutations. Functional platelet studies however showed an obvious Gs hypofunction in the 13 patients that were available for testing. Methylation for the three differentially methylated GNAS regions was quantified via the Sequenom EpiTYPER. Patients showed significant hypermethylation of the XL amplicon compared to controls (36±3 vs. 29±3%; p

Despite compelling epidemiological evidence that folic acid supplements reduce the frequency of neural tube defects (NTDs) in newborns, common variant association studies with folate metabolism genes have failed to explain the majority of NTD risk. The contribution of rare alleles as well as genetic interactions within the folate pathway have not been extensively studied in the context of NTDs. Thus, we sequenced the exons in 31 folate-related genes in a 480-member NTD case-control population to identify the full spectrum of allelic variation and determine whether rare alleles or obvious genetic interactions within this pathway affect NTD risk. We constructed a pathway model, predetermined independent of the data, which grouped genes into coherent sets reflecting the distinct metabolic compartments in the folate/one-carbon pathway (purine synthesis, pyrimidine synthesis, and homocysteine recycling to methionine). By integrating multiple variants based on these groupings, we uncovered two provocative, complex genetic risk signatures. Interestingly, these signatures differed by race/ethnicity: a Hispanic risk profile pointed to alterations in purine biosynthesis, whereas that in non-Hispanic whites implicated homocysteine metabolism. In contrast, parallel analyses that focused on individual alleles, or individual genes, as the units by which to assign risk revealed no compelling associations. These results suggest that the ability to layer pathway relationships onto clinical variant data can be uniquely informative for identifying genetic risk as well as for generating mechanistic hypotheses. Furthermore, the identification of ethnic-specific risk signatures for spina bifida resonated with epidemiological data suggesting that the underlying pathogenesis may differ between Hispanic and non-Hispanic groups.