Analysis of Differences in DNA Damage and Repair Efficacy in Lymphocytes of Patients with Bipolar Disorder. / Bipolar Bozukluk Tanili Hastalarin Lenfositlerinde DNA Hasari ve Tamir Etkinligindeki Farkliliklarin Incelenmesi.

OBJECTIVE: The aim of this study was to determine DNA damage during euthymic and attack periods, and the oxidative metabolism states that may cause this damage in the pathophysiology of bipolar disorder. The role of DNA repair mechanisms in this process was also investigated. METHOD: The study included a total of 90 patients aged between 18-65 years who were diagnosed with bipolar disorder according to DSM- 5 diagnostic criteria, with 30 patients in euthymic, 30 in manic and 30 in depressive periods. A control group was formed of 30 healthy subjects matched to the patients by age, gender, body mass index and smoking status and/or alcohol consumption. Oxidative metabolism was investigated using the Comet Assay technique to assess DNA damage, according to the oxidant/antioxidant status in the technique developed by Erel with the Rel ASSAY Diagnostics kit (Turkey). The control and patient groups were compared in respect of gene expression levels of OGG1 and NEIL1 repair genes at mRNA level with Real-Time PCR. RESULTS: Increased DNA damage was found in the euthymic and manic groups and decreased NEIL1 gene expression in the depressive group. The oxidative stress index was found to be decreased in the patient groups compared to the healthy control group. CONCLUSION: Oxidative imbalance and DNA damage and repair disorders may be effective in the pathophysiology of bipolar disorder. Further studies on this subject are required to clarify the etiology and new treatment goals.

The high frequency of chromosomal copy number variations and candidate genes in epilepsy patients.

OBJECTIVE: Epilepsy is a chronic brain disease and is estimated to affect more than 50 million people worldwide.Epilepsy is a polygenic and multifactorial disease.Genetic causes play a major role in 40-60 % of all epilepsies.Copy number variations(CNVs) have been reported in approximately 5-12 % of patients with different types of epilepsy.Here we aimed to determine the diagnostic yield of the aCGH in epilepsy and to reveal new candidate genes and CNVs by analyzing aCGH data retrospectively. METHODS: The clinical data of 80 patients with the diagnosis of epilepsy were examined retrospectively and the raw data of aCGH of these patients were reanalyzed in the light of current literature. RESULTS: Pathogenic/likely pathogenic CNVs were detected in 14 of 80 patients and 12 of these CNVs (15 %) were associated with epilepsy phenotype. In addition, 18 CNVs in 16 different chromosomal loci that were evaluated as the variant of unknown clinical significance(VOUS). In four cases (5%), CNVs associated with epilepsy were less than 100 kb and these accounted for 13.3 % of all epilepsy associated CNVs. CONCLUSION: The diagnostic yield of aCGH in epilepsy patients was found to be higher than most studies in the literature. MACROD2,ADGRB3(BAI3),SOX8,HIP1,PARK2 and TAFA2 genes were evaluated as potential epilepsy-related genes and NEDD9,RASAL2 and TNR genes thought to be the candidate genes for epilepsy. Our study showed that the diagnostic efficiency of aCGH in epilepsy is high and with more comprehensive studies, it will contribute to the elucidation of genes involved in genetic etiology in epilepsy patients.

Blau syndrome with a rare mutation in exon 9 of NOD2 gene.

Blau syndrome is an autosomal dominant rare disease caused by mutations in NOD2 gene. Less than 200 patients published with Blau Syndrome Worldwide. We reported a 41-year old female Turkish patient diagnosed as Blau syndrome. Granulomatous dermatitis and severe headache, as well as recurrent chest and pelvic pain have been present since she was 8 years old. Arthritis started when she was teenage, hypertension diagnosed when she was 20 and other symptoms also occurred during the lifetime (severe preeclampsia, ischemic stroke, recurrent hemiparesis, recurrent-transient-vision-loss and renal-artery-stenosis). Genomic DNA was isolated from peripheral blood and 12 genes sequenced in Autoinflammatory panel on IonTorrent-S5-NGS platform with Parseq-VariFind™AIPassay. NGS analysis showed 107 variants in in the index case, mainly benign with no strong association with Blau syndrome. Additionally, we identified one very rare missense mutation in NOD2 gene (c2803G>A, p.Val935Met) and in silico assessment of the mutation indicated possible pathogenic significance and strong association with Blau syndrome. In addition, we analyzed family members of the index case and identified the same mutation in NOD2 gene. The segregation analysis shows the presence of the same mutant allele in NOD2 gene in the index case affected sister, as well as in her son with arthralgia, while in her non affecter brother we didn't detect the Val935Met mutation in NOD2 gene. Blau Syndrome is known as a very rare disease, mainly caused by mutations in NOD2 gene. Missense mutation diagnosed in our case could be responsible for the phenotype of the index case. Our results indicate the importance of NGS testing and its major role in the detection of rare mutations that may responsible for the onset of autoinflammatory disorders.

De novo mutations of the ATP6V1A gene cause developmental encephalopathy with epilepsy.

V-type proton (H+) ATPase (v-ATPase) is a multi-subunit proton pump that regulates pH homeostasis in all eukaryotic cells; in neurons, v-ATPase plays additional and unique roles in synapse function. Through whole exome sequencing, we identified de novo heterozygous mutations (p.Pro27Arg, p.Asp100Tyr, p.Asp349Asn, p.Asp371Gly) in ATP6V1A, encoding the A subunit of v-ATPase, in four patients with developmental encephalopathy with epilepsy. Early manifestations, observed in all patients, were developmental delay and febrile seizures, evolving to encephalopathy with profound delay, hypotonic/dyskinetic quadriparesis and intractable multiple seizure types in two patients (p.Pro27Arg, p.Asp100Tyr), and to moderate delay with milder epilepsy in the other two (p.Asp349Asn, p.Asp371Gly). Modelling performed on the available prokaryotic and eukaryotic structures of v-ATPase predicted p.Pro27Arg to perturb subunit interaction, p.Asp100Tyr to cause steric hindrance and destabilize protein folding, p.Asp349Asn to affect the catalytic function and p.Asp371Gly to impair the rotation process, necessary for proton transport. We addressed the impact of p.Asp349Asn and p.Asp100Tyr mutations on ATP6V1A expression and function by analysing ATP6V1A-overexpressing HEK293T cells and patients' lymphoblasts. The p.Asp100Tyr mutant was characterized by reduced expression due to increased degradation. Conversely, no decrease in expression and clearance was observed for p.Asp349Asn. In HEK293T cells overexpressing either pathogenic or control variants, p.Asp349Asn significantly increased LysoTracker® fluorescence with no effects on EEA1 and LAMP1 expression. Conversely, p.Asp100Tyr decreased both LysoTracker® fluorescence and LAMP1 levels, leaving EEA1 expression unaffected. Both mutations decreased v-ATPase recruitment to autophagosomes, with no major impact on autophagy. Experiments performed on patients' lymphoblasts using the LysoSensor™ probe revealed lower pH of endocytic organelles for p.Asp349Asn and a reduced expression of LAMP1 with no effect on the pH for p.Asp100Tyr. These data demonstrate gain of function for p.Asp349Asn characterized by an increased proton pumping in intracellular organelles, and loss of function for p.Asp100Tyr with decreased expression of ATP6V1A and reduced levels of lysosomal markers. We expressed p.Asp349Asn and p.Asp100Tyr in rat hippocampal neurons and confirmed significant and opposite effects in lysosomal labelling. However, both mutations caused a similar defect in neurite elongation accompanied by loss of excitatory inputs, revealing that altered lysosomal homeostasis markedly affects neurite development and synaptic connectivity. This study provides evidence that de novo heterozygous ATP6V1A mutations cause a developmental encephalopathy with a pathomechanism that involves perturbations of lysosomal homeostasis and neuronal connectivity, uncovering a novel role for v-ATPase in neuronal development.

Congenital Central Hypothyroidism Caused by a Novel Thyroid-Stimulating Hormone-Beta Subunit Gene Mutation in Two Siblings.

Congenital central hypothyroidism (CCH) is a very rare disease. Alterations in pituitary development genes as well as mutations of immunoglobulin superfamily member 1 and transducin ß-like protein 1 can result in CCH and multiple pituitary hormone deficiencies. However, mutations of the thyrotropin-releasing hormone receptor or thyroid-stimulating hormone-beta (TSHB) gene are responsible for isolated CCH. In this paper, we present the cases of two siblings with a novel mutation of TSHB. Direct sequencing of the coding regions and exon/intron boundaries of the TSHB gene revealed two homozygous nucleotides changes. One of them was c.40A>G (rs10776792) which is a very common variation that is also seen in healthy individuals, the other was c.94G>A at codon 32 of exon 2 which resulted in a change from glutamic acid to lysine (p.E32K). Both patients were homozygous and the parents were heterozygous.