Moleküler Biyoloji ve Genetik

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Aberrant global DNA methylation in neurodegeneration: ALS and trinucleotide repeat disorders
(Thesis (M.S.) - Bogazici University. Institute for Graduate Studies in Science and Engineering, 2016., 2016.) Hamzeiy, Hamid.; Battaloğlu, Esra.; Başak, A. Nazlı
Amyotrophic lateral sclerosis (ALS) is a late onset neurodegenerative disease of the motor neurons, leading to death within two-three years of onset. Extensive studies have thus far helped identify many genetic causes for this devastating disease, but despite such efforts, more than 80% of ALS still remains unexplained. ALS is generally considered to be a polygenic disease, and many different factors may be involved in its pathogenesis, including a combination of rare mutations and environmental factors which could ultimately lead to epigenetic modifications. Here, we investigated ALS from an epigenetic perspective, focusing on 5-methylcytosine (5-mC), a well-characterized epigenetic modification, aiming to conclude disputes between different studies reporting inconsistent results for global 5-mC levels detected in blood samples of sporadic ALS (sALS) patients. The study was further extended to different subtypes of ALS, including familial ALS (fALS), C9orf72 expansion carrier ALS (C9orf72+ ALS) and ATXN2 intermediate expansion ALS along with spinocerebellar ataxia types 1 and 2 (SCA1 and SCA2), Huntington’s disease, Friedreich’s ataxia and myotonic dystrophy type 1. In order to analyze the global 5-mC levels, in DNA isolated from blood, an enzyme-linked immunosorbent assay (ELISA) kit was selected upon testing of commercially available kits. The results showed that increased global 5-mC levels are not exclusive to sALS (p < 0.001 [F(1, 214) = 11.993, p = 0.000645]) and that this can also be observed in different subtypes of fALS. Interestingly, SCA1 (p < 0.01 [F(1, 32) = 8.778), p = 0.00571]) and SCA2 (p < 0.01 [F(1, 56) = 10.784, p = 0.001768]) patients also showed increased levels of global 5-mC when compared to age- and sex-matched healthy controls. Additionally, direct bisulfite sequencing was utilized to investigate the C9orf72 promoter in C9orf72+ ALS patients and healthy controls. Promoter hypermethylation was observed in patients and was moderately correlated (rs = 0.3902, p < 0.05) with the global levels of 5-mC. We also tested several commercial kits for the quantification of 5-hydroxymethylcytosine and could not find a suitable kit for its detection in blood.
Amyotrphic lateral sclerosis in Turkey : Studies on Familial and sporadic ALS using high-throughput genomic technologies
(Thesis (Ph.D.)-Bogazici University. Institute for Graduate Studies in Science and Engineering, 2010., 2010.) Özoğuz, Aslıhan.; Başak, A. Nazlı.
Amyotrophic Lateral Sclerosis (ALS) is a late-onset neurodegenerative disease, characterized by death of motor neurons in cortex, brainstem and spinal cord. It is a multifactorial disease with interacting pathogenic mechanisms. Most incidences are sporadic (SALS), while 10 per cent of cases have a family history (FALS). The genetics of ALS is complex; eight genes and six loci with autosomal dominant (AD), autosomal recessive and X-linked patterns of inheritance have been identified thus far. In the framework of this study, 198 Turkish ALS cases were investigated for possible mutations in the SOD1 gene. Five FALS cases were shown to carry disease-causing SOD1 mutations, while six were carriers of a rare polymorphism. In the next step, AD, nonconsanguineous FALS and juvenile cases were analyzed for the TDP-43, FUS and ANG genes via DNA sequencing. One homozygous D90A case, represented as recessive, was investigated by haplotype analysis and was compared to 21 Scandinavian ALS cases in search of a common ancestry. Additionally, 15 FALS and 13 juvenile cases, who were negative for the tested genes, were analyzed by whole genome genotyping for identification of new ALS genes. While no significant regions were obtained, a recessive family was preselected for the identification of homozygosity regions. Five candidate genes located within homozosity regions were examined in one of the family member; no mutations were identified. The same individual was also assessed by whole exome resequencing. Furthermore, this study also contributed to a collaborative genome-wide association study in SALS, where 14 month-survival advantage was shown for homozygous CC allele at rs1541160 SNP in the gene coding KIFAP3. This is the most comprehensive study performed in Turkey on the molecular genetics of ALS. The high-throughput methodologies used and the findings presented in this thesis are expected to shed light to the complex pathogenesis of amyotrophic lateral sclerosis.
Analysis of the vascular endothelial growth factor and angiogenin genes as risk factors for amyotrophic lateral sclerosis in the Turkish population; identification of a possible novel mutation
(Thesis (M.S.)-Bogazici University. Institute for Graduate Studies in Science and Engineering, 2006., 2006.) Güzel, Refika Mine.; Başak, A. Nazlı.
Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disorder, which is primarily characterized by the death of motor neurons in the cerebral cortex, spinal cord, and brainstem. Degeneration of motor neurons leads to progressive wasting and eventual paralysis of skeletal muscles. Only a small portion of all ALS cases show familial inheritance (FALS), and the remaining majority of patients are sporadic (SALS). Still, a genetic component is thought to contribute to SALS pathogenesis. Despite intensive research, the mechanisms leading to neurodegeneration in ALS have not been fully understood. Identification of genes, either underlying ALS or predisposing to ALS, has been an important part of ALS research. Among several etiologic gene candidates, thought to be associated with SALS, Vascular Endothelial Growth Factor (VEGF) and Angiogenin (ANG) aroused particular interest, because they are well-known factors for their angiogenic activities. In the framework of this thesis, we examined the link between ALS and the reported VEGF and ANG gene variations in the Turkish population, since it is very important to confirm the observed genetic association in various different populations. Screening of 101 ALS patients and 99 healthy controls with restriction enzyme analysis and DNA sequencing did not reveal any statistically significant association of these genes with ALS in our study population. But a possible novel mutation in the ANG gene was identified in a juvenile ALS patient. This is a preliminary result, which has to be verified by further analysis of the patient and his family.
Genetic factors contributing to ALS in Turkey: ATXN2 polyQ repeat expansions and SNP/CNV associations
(Thesis (M.S.) - Bogazici University. Institute for Graduate Studies in Science and Engineering, 2013., 2013.) Ömür, Özgül.; Başak, A. Nazlı.
ALS is the most frequent motor neuron disorder and so far no cure has been developed for this devastating disease. ALS is characterized by the selective loss of both the upper motor neurons of the motor cortex and the lower motor neurons extending through the brainstem and spinal cord to skeletal muscle. The majority of ALS patients (86.5-99 per cent) are sporadic and the remaining cases have a reported family history. With more than 25 genes and several loci identified so far, approximately 60 percent of familial and only a small proportion of sporadic ALS cases have been explained. Thus, identification of new genes contributing to disease is crucial. A comprehensive study combining yeast screen, fly/cell biology and human genetics led to the identification of the association of intermediate length (27-33) PolyQ expansions in the ATXN2 gene and ALS risk. In this study, the distribution of ATXN2 PolyQ sizes in a Turkish cohort including 236 patients and 420 healthy controls were investigated. Furthermore, to evaluate other possible genetic associations of this locus with ALS, SNP and haplotypes were analyzed. In addition to SNP and haplotype analyses, validation of candidate CNVs in the MAP4K3, HLA-B and EPHA3 genes, previously found to be disease-associated, was performed. In accordance with other studies, this thesis confirmed that >30 PolyQ repeats in the ATXN2 gene are associated with ALS risk in 1.7% of the Turkish ALS cohort under study. Additionally, a significant association of a 5-SNP haplotype block across the ATXN2 and SH2B3 genes was found in three per cent of the ALS cohort, which was not present in the controls (p = 0.0011). CNV validation studies revealed that EPHA3 is a possible protective factor against ALS in the Turkish ALS cohort under study. EPHA3 is one of the members of receptor tyrosine kinase (RTK) family and ATXN2 and SH2B3 encode proteins that both interact with growth receptor tyrosine kinases. Our novel observation suggests that proteins of RTK pathway and their interaction partners may be promising targets for therapeutic interventions.
Genetically accurate models of SOD1-based ALS in drosophila: validation and characterization
(Thesis (M.S.) - Bogazici University. Institute for Graduate Studies in Science and Engineering, 2013., 2013.) İskender, Ceren.; Başak, A. Nazlı.
SOD1 has been a unique target of ALS research as the first identified gene of the disease; since 20 years, SOD1 has not been related to any other disease unlike other ALScausing genes. There are more than 160 mutations seen in ALS patients scattered all around the small protein that cause the mutant protein to gain a toxic function. Up to date, more than 20 SOD1-based animal models have been developed that helped us gain insights to the mechanisms in which mutant SOD1 is involved, e.g. oxidative stress, misfolded protein aggregation, endoplasmic reticulum stress and glutamate excitotoxicity. Even so, all these models had their own drawbacks as being either knock-out or over-expression studies. In this respect we aimed to develop an accurate model of SOD1-based ALS using ends-out homologous recombination (HR) in Drosophila. In this study, four previously defined SOD1 missense mutations (G37R, H48R, H71Y and G85R) were introduced into the endogenous locus of Drosophila SOD1 (dSOD). For the characterization of mutants, life span and larval motility assays were used. In addition, genotypic ratios of the mutants were observed for genotype-phenotype correlations and expression levels of several candidate genes, possibly involved in ALS pathogenesis, were compared among mutants using quantitative RT-PCR. This is the first study, which successfully implements homologous recombination, a new and powerful approach to ALS. We hope that, genetically accurate models of SOD1-based ALS, generated via homologous recombination, will further help us gain insights into the complex mechanisms involved in neurodegenerative processes.
Investigation of motor neuron diseases by wes: genetic dissection of a Turkish ALS cohort
(Thesis (M.S.) - Bogazici University. Institute for Graduate Studies in Science and Engineering, 2017., 2017.) Akçimen, Fulya.; Battaloğlu, Esra.; Başak, A. Nazlı.
Amyotrophic lateral sclerosis (ALS), the most common motor neuron disease, is characterized by muscle weakness and atrophy due to the degeneration of motor neurons in the motor cortex, brain stem and spinal cord. Both conventional gene discovery methods and association studies helped identify the genetic variants causing several ALS phenotypes. Recently, with the advent of whole exome sequencing (WES), it became possible to sequence the coding regions of the genome for a low cost and in a short time, changing the landscape of genetic disease research, including ALS. Thus, there are more than 40 genes with Mendelian inheritance identified in ALS. However, a significant portion of ALS cases is still genetically unexplained due to the complex genetic background of the disease. In this study, WES was applied to investigate disease-causing variants in a cohort of 57 cases with ALS or other motor neuron diseases. In silico workflow was performed in our laboratory from the raw sequencing data to the final candidate variant lists. Homozygosity mapping was applied to recessively inherited pedigrees. Mutations in 19 distinct genes were identified as the genetic cause in 20 families. Identification of genes causing distal spinal muscular atrophy and neurodegeneration with brain iron accumulation in some cases, suggested controversies between the initial and the final diagnosis of the patients. These findings allowed us to draw two main facts: (i) the complex and heterogeneous nature of ALS and other motor-neuron diseases due to phenotypic overlaps, and (ii) the great success of WES as a current trend in rare disease genetics and differential diagnosis.
Investigation of the effects of SUMO modification on two critical proteins : a pathogenic NEK1 mutant that drives ALS pathogenesis and the CRISPR-associated CAS9 protein
(Thesis (M.S.) - Bogazici University. Institute for Graduate Studies in Science and Engineering, 2022., 2022) Ergünay, Tunahan.; Şahin, Umut.
Small Ubiquitin-like modifier (SUMO) is an essential eukaryotic post-translational modification. SUMO isoforms attach to specific lysine residues on target substrates to modi-fy their function, activity, solubility and stability. Dysregulation of sumoylation is associated with various pathological conditions ranging from cancer to neurodegeneration. In this study, we focused on a mutant form of NEK1 protein, called truncated NEK1 (or tNEK1), which was recently linked to the pathogenesis of Amyotrophic Lateral Sclerosis (ALS). Previous studies from our lab had established that tNEK1 was prone to aggregation and associated with PML nuclear bodies (NBs). Here, we showed that PML also facilitates tNEK1 sumoylation and ubiquitylation. Furthermore, pharmaceutical agents that induce PML NB biogenesis, such as interferon-alpha (IFN), promote tNEK1 hypersumoylation in a PML-dependent manner. These findings have important implications for the management of tNEK1-linked ALS in the clinic, as IFN may promote the hypersumoylation, degradation, and clearance of this toxic protein in vivo, in a PML-dependent manner. In addition, our lab has created a transgenic mice model that expressed tNEK1 to study the effect of this mutation in vivo. In this study, we have performed motor neuron function assays (specifically, foot-print and walking assays). Our results indicate that tNEK1-expressing mice display a walk-ing disorder, implying that tNEK1 expression in vivo may lead to ALS pathogenesis. Finally, we have also conducted studies on the CRISPR-associated Cas9 protein that we have recently discovered to be a sumoylation target. We have identified the major SUMO conjugation site on this protein and showed that sumoylation impacted on both the stability and DNA-binding ability of this important protein.
Investigation of the pathogenic basıs of NEK1-linked amyotrophic lateral sclerosis and discovery of a potential treatment that prevents protein aggregation through targeted destruction
(Thesis (M.S.) - Bogazici University. Institute for Graduate Studies in Science and Engineering, 2019., 2019.) Öztürk, Harun.; Şahin, Umut.
Amyotrophic lateral sclerosis (ALS) is a debilitating neurodegenerative disease with dismal prognosis, characterized by motor neuron loss in the patients. More than 30 genes have been reported to be involved in ALS pathogenesis. Most recently, re searchers identiﬁed novel mutations on NEK1, a kinase that functions in several pro cesses including cell cycle control and early DNA damage response. However, how mutant NEK1 drives ALS pathogenesis is hitherto unknown. Here we reveal that a NEK1 variant commonly found in ALS patients, truncated NEK1 (tNEK1), displays altered subcellular localization and is trapped in the nucleus due to the loss of two nuclear export signals in the mutant form. We show that tNEK1 displays massive loss of solubility, forming dramatic intracellular aggregates, a phenomenon commonly seen in most ALS cases. Nuclear tNEK1 is recruited in Promyelocytic Leukemia (PML) Nuclear Bodies (NBs) which act as catalytic chambers that promote Ubiquitin-like modiﬁcations and initiate protein degradation; consistent with this, we found that tNEK1 undergoes massive modiﬁcation by Small Ubiquitin-Like Modiﬁer (SUMO). Drugs that enhance PML NB biogenesis and function, such as interferon alpha (IFN), promote tNEK1 recruitment in PML NB, its post-translational modiﬁcations and sub sequent destruction by the proteasome, likely before it gets a chance to aggregate. In conclusion, in our study we shed light on how a NEK1 mutant might drive ALS pathogenesis, and identify IFN as a potential treatment for tNEK1-linked ALS cases.
Possible interactors of alsin and effects of its expression on four motor neuron disease-related proteins
(Thesis (M.S.) - Bogazici University. Institute for Graduate Studies in Science and Engineering, 2012., 2012.) Çobanoğlu, Gönenç.; Başak, A. Nazlı.
Motor neuron disorders, characterized by selective motor neuron degeneration, consist of a large group of diseases with overlapping genes and common pathological mechanisms. Mutations in ALS2, associated with three types of motor neuron disorders with early age of onset, result in a loss of function of the protein, suggesting a crucial role of the protein product, alsin, in motor neuron integrity. However, the exact function of alsin is not known yet. This study aims to gain insights into alsin’s function by investigating its relations both at mRNA and protein levels with proteins that are involved in different motor neuron disorders. Spastin (Spg4) and spartin (Spg20), selected as representative genes, are involved in upper motor neuron disorders, while heat shock protein B1 (HspB1) and heat shock protein B8 (HspB8) were selected due to their associations with lower motor neuron disorders. Since ALS2 mutations result in a loss of function, an Als2 knock-down stable cell line was generated. Q-RT PCR revealed statistically significant alterations in the expression levels of Sgp20, Spg4, HspB1 and HspB8 in this cell line, as compared to the control cell line. For the first time in literature, the inhibitory effect of Als2 on HspB1 expression under heat shock conditions implicated a possible role of alsin in heat shock response. At the protein level, it was observed that spartin and alsin were colocalized in the perinuclear region of differentiated neuronal N2a cells. Furthermore, immunoprecipitation studies revealed the co-precipitation of these proteins in the same protein complex, indicating a possible physical interaction. We hope that this thesis, the first study, showing relations of alsin with spartin and HspB1, will contribute to this unexplored field.
The assessment of ubiquitin E3 ligase activity of RNF4 on ALS-related NEK1 protein
(Thesis (M.S.) - Bogazici University. Institute for Graduate Studies in Science and Engineering, 2019., 2019.) Canaslan, Sezgi; Şahin, Umut.
Amyotropic lateral sclerosis (ALS) is a serious and progressive motor-neuron disease in which neurons that control voluntary muscles gradually deteriorate. Patients lose the ability to speak, move and breathe. To date, 30 ALS associated genes have been discovered. The products of mutated genes generally follow the similar pattern which is protein aggregation. In 2014, the results of the global MinE project have announced that NEK1 is a new ALS-related gene. NEK1 is a serine/threonine kinase functioning in cell cycle regulation, DNA damage response, cilia formation, etc. However, its contribution to disease phenotype is still not known. If NEK1 shares the same characteristic with other ALS-related proteins, it tends to aggregate. These aggregates can be cleared with the help of ubiquitin-proteasome system. In this study, we investigated whether NEK1 is a new target for RNF4 which is a special ubiquitin E3 ligase. If RNF4 is an E3 ligase for NEK1, it may affect the stability of NEK1 leading to its degradation by the proteasome. In silico anaylsis and co-immunoprecipitation experiments have suggested that NEK1 and RNF4 physically interact. RNF4 overexpression changes the stability of NEK1. In addition, the silencing of RNF4 with siRNA causes also the stabilization of NEK1. We have analyzed the ubiquitination of wild type NEK1 and one of its the disease-related versions, tNEK1. According to our preliminary results, NEK1 is ubiquitinated and tNEK is hyperubiquitinated. In conclusion, our study shows that NEK1 can be a new substrate of RNF4 and RNF4 can lead to further ubiquitination of NEK1 and tNEK1. These findings can contribute to improvements in therapeutic interventions for still incurable ALS.
The genetics of sporadic ALS: the first Turkish GWAS and novel SNP and CNV association
(Thesis (M.S.) - Bogazici University. Institute for Graduate Studies in Science and Engineering, 2012., 2012.) Uyan, Özgün.; Başak, A. Nazlı.; Özçelik, Hilmi.
Selective degeneration of both lower and upper motor neurons located in the brain cortex, brainstem and ventral horn of the spinal cord result in amyotrophic lateral sclerosis (ALS). ALS is the most frequent late-onset motor neuron disorder and so far no cure or treatment for ALS has been developed. The vast majority of ALS cases are sporadic, approximately 10% of patients have a family history. At least 25 genes and several loci have been identified so far in familial cases. The genetic factors that contribute to ALS correspond to atmost 10% in sporadic cases so there is a huge genetic gap in sALS. The missing part of genetic factors needs to be revealed to fill the lacking players in the mechanisms underlying disease pathogenesis. GWAS enables large scale cohort studies between cases and controls to pinpoint new candidate genomic regions that may be associated with disease phenotype. In the framework of this thesis, we aimed to investigate sporadic ALS patients to identify novel Single Nucleotide Polymorphisms (SNPs), Copy Number Variations (CNVs) and genes, associated with ALS, using GWAS and computational biology approaches. First, we analyzed single marker associations of SNPs in the regions of fALS-causing genes in two published datasets. The ACA haplotype in TARDBP locus was found to be associated with ALS. Next, we performed GWAS on Turkish ALS samples, which included 116 sALS patients and 109 controls. We identified two SNP clusters in TMEM90B and CPNE5 genes associated with ALS, yet all SNPs failed to keep the significance after Bonferroni correction. Third, we performed genome-wide CNV analysis using raw data of GWAS and the PennCNV tool. We identified several novel and reported CNVs in gene and intergenic regions. To the best of our knowledge, this is the first ALS-GWAS of sALS patients in Turkey. We hope that the findings of this thesis in first-stage GWAS and genome-wide CNV analyses will shed light to the unknown genetics of sALS and help to pave the ways in the identification of novel genes associated with sALS pathogenesis.
The impact of mutant alsin protein on motor neuron models and the effects of alsin gene knock-down on motor protein genes
(Thesis (M.S.)-Bogazici University. Institute for Graduate Studies in Science and Engineering, 2011., 2011.) Lahut, Suna.; Başak, A. Nazlı.
Alsin is a ubiquitously expressed protein with the highest levels in the central nervous system. Mutations in the alsin gene have been associated with several juvenile onset recessive motor neuron diseases. Nine out of 12 reported mutations in the alsin gene are deleterious mutations, leading to premature termination of the protein by an early stop codon; all affected patients are homozygous and all unaffected siblings are heterozygous or normal for the alsin mutations. These together, suggest a loss of function mechanism, responsible for motor neuron degeneration. The aim of this study is to understand the function of alsin using two different approaches. The first approach investigates the effects of mutant alsin on cells with motor neuron characteristics. The alsin mutation generated in this study leads to the truncation of one third of the protein, including the VPS domain, as it is the case in the majority of the natural mutations. The second approach analyzes alsin’s possible interactions with six motor proteins, which have a role in axonal transport: DCTN1, DCTN2, DCTN3, KIF3A, KIF3B and KIF5A. The results of this thesis suggest a rapid degradation of the mutant alsin protein in the NSC34 cells. Thus, it concludes that the pathogenesis of ALS might be due to the overall loss of the alsin protein rather than the loss of the VPS domain, as reported before in the literature. This study also implicates a possible interaction of alsin with three of the six investigated proteins: DCTN1, KIF3A and KIF3B. It further shows a co-localization of alsin and KIF3A in the perinuclear region of N2a cells. This thesis is a first attempt in our laboratory for elucidating the alsin-interacting partners in cell culture systems; we hope that this study will help to pave the ways for understanding the complicated and multilayered functions of the alsin protein.
The molecular landscape of ALS in Turkey: A multifaceted approach to the complex genetics of ALS
(Thesis (Ph.D.)-Bogazici University. Institute for Graduate Studies in Science and Engineering, 2018., 2018.) İskender, Ceren.; Çağlayan, S. Hande.; Başak, A. Nazlı.
Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease (NDD), characterized by degeneration of both upper and lower motor neurons leading to muscle wasting. With an incidence of two and a prevalence of four in 100.000/year, it is the third most common NDD after Alzheimer’s and Parkinson’s diseases. The last decade has proven that the ‘condition’ called ALS is both clinically and genetically heterogenous, and that the genetic component in 90% of the cases that are considered as sporadic, might be stronger than expected. In this thesis, we investigated the complex genetics of ALS in the ethnically heterogeneous yet inbred Turkish population. Familial ALS account for 22% of our cases (159/722) and 563 cases were classified as sporadic (78%). Consanguinity is calculated as 25% among familial and 17% in sporadic cases. Conventional screening of the most common ALS genes (C9orf72, SOD1, TARDBP and FUS) in patients with a family history of disease, explained the disease cause in only 33%, pointing towards marked locus heterogeneity within the population. C9orf72 hexanucleotide repeat expansion was further detected in 3% of ‘apparently sporadic’ patients. Application of whole exome sequencing (WES) in dominant and recessive Turkish pedigrees presenting with ALS or non-ALS motor neuron diseases (MNDs), revealed distinct rare or novel mutations in 20 out of 39 families and enabled differential diagnosis in cases with atypical ALS features. Common current themes in ALS, like oligogenic inheritance and possible genetic modifiers are addressed in families with incomplete penetrance and in C9orf72 expansion carriers. Oligogenic inheritance of known ALS genes was not prominent in the Turkish C9orf72-positive cases. Hypermethylation in the promoter region of C9orf72 was confirmed in Turkish patients, however it did not seem to modify age of onset. Finally, analyses of whole genome sequencing data of 625 Turkish ALS patients and 152 healthy controls in the framework of Project MinE showed population-specific aspects and once more substantiated the concept of low penetrance of rare ALS genes among sporadic patients.

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